In vitro Developmental Competence of Adult Sheep Oocytes Treated with Roscovitine.

The efficiency of in vitro sheep embryo production is still low compared to that observed in vivo and in other species. In this context, meiotic inhibition strategies emerged as a promising alternative to improve this biotechnology. So, this study aimed to evaluate, for the first time, the effects of roscovitine on in vitro maturation of sheep oocytes and their subsequent embryo development. For this, cumulus-oocyte complexes (COCs) were cultured for 6 h in the presence (Rosco) or absence (Control) of 75 µm roscovitine and, subsequently, in vitro matured (IVM) for 18 h with gonadotropins. At 0 (Immature), 6 and 24 h of culture, the nuclear status of oocytes was evaluated by Hoechst staining. Embryo cleavage and blastocyst formation were recorded 30 h after in vitro fertilization and on day 7 of culture, respectively. Blastocyst quality was evaluated by differential staining. At 6 h, the GV rate in the Rosco treatment (93.8%) was similar to that observed in the Immature oocytes (94.9%) and significantly higher compared to Control (41.3%). After IVM for 18 h, a high and similar proportion of oocytes from Rosco (93.6%) and Control (88.4%) reached the MII stage. In both treatments, approximately 70% of oocytes cleaved and 50% of them developed up to blastocyst. The mean percentage of blastocyst cells, embryoblast, trophoblast and pyknosis did also not differ between Control and Rosco. In conclusion, roscovitine, at the studied experimental conditions, was efficient to reversibly inhibit the meiosis of adult sheep oocytes without detrimental effect on development and quality of the in vitro produced embryos.

Effect of oil overlay on inhibition potential of roscovitine in sheep cumulus-oocyte complexes.

Inhibitors of cyclin-dependent kinases, as roscovitine, have been used to prevent the spontaneous resumption of meiosis in vitro and to improve the oocyte developmental competence. In this study, the interference of oil overlay on the reversible arrest capacity of roscovitine in sheep oocytes as well as its effects on cumulus expansion was evaluated. For this, cumulus-oocyte complexes (COCs) were cultured for 20 h in TCM 199 with 10% foetal bovine serum (Control) containing 75 µm roscovitine (Rosco). Subsequently, they were in vitro matured (IVM) for further 18 h in inhibitor-free medium with LH and FSH. The culture was performed in Petri dishes under mineral oil (+) or in 96 well plates without oil overlay (-) at 38.5°C and 5% CO2 . At 20 and 38 h, the cumulus expansion and nuclear maturation were evaluated under stereomicroscope and by Hoechst 33342 staining, respectively. No group presented cumulus expansion at 20 h. After additional culture with gonadotrophins, a significant rate of COCs from both Control groups (+/-) exhibited total expansion while in both Rosco groups (+/-) the partial expansion prevailed. Among the oocytes treated with roscovitine, 65.2% were kept at GV in the absence of oil overlay while 40.6% of them reached MII under oil cover (p < 0.05). This meiotic arrest was reversible, and proper meiosis progression also occurred in the Control groups (+/-). So, the culture system without oil overlay improved the meiotic inhibition promoted by roscovitine without affecting the cumulus expansion rate or the subsequent meiosis progression.

Controle molecular do acúmulo, tradução e degradação de mRNA em oócitos e embriões / Molecular control of accumulation, translation and degradation of mRNA in oocytes and embryos

A competência oocitária para suportar os posteriores estágios do desenvolvimento depende não somenteda correta segregação cromossômica e de transformações citoesqueléticas, mas, principalmente, da adequadatranscrição e estoque de mRNAs cruciais ao desenvolvimento e à viabilidade celular. Com a retomada dameiose, no entanto, embora o oócito mantenha a capacidade de tradução gênica e de síntese proteica, suaatividade transcricional é interrompida, sendo restabelecida apenas com a ativação do genoma embrionário.Deste modo, todo mRNA materno mobilizado durante maturação, expansão do cumulus, fertilização eembriogênese inicial deve ser sintetizado e estocado, em sua forma traducionalmente inativa, nos oócitosmantidos no estágio diplóteno da prófase I. Complexos mecanismos regulatórios, os quais envolvem apoliadenilação e a desadenilação do mRNA, estão implicados no processo de ativação e silenciamento tantotranscricional quanto traducional. Assim, dada à relevância do tema, esta revisão se propõe a abordar osprincipais eventos moleculares envolvidos no controle da expressão, do estoque, da tradução e da degradação detranscritos maternos imprescindíveis ao desenvolvimento oocitário e embrionário.

The oocyte competence to support the later stages of development depends not only on correctchromosome segregation and cytoskeletal changes, but mainly, the proper transcription and storage of mRNAscritical to the cellular development and viability. However, with the resumption of meiosis, although the oocytekept the ability of gene translation and protein synthesis, its transcriptional activity is interrupted and restoredonly with embryonic genome activation. Thus, all maternal mRNA mobilized during maturation, cumulusexpansion, fertilization and early embryogenesis must be synthesized and stored, in its translationally inactiveform, in oocytes kept in the diplotene stage of prophase I. Complex regulatory mechanisms which involvedeadenylation and polyadenylation of mRNA are involved in this process of activation and silencing astranscriptional as translational. So, due to the importance of the topic, this review proposes to discuss the mainmolecular events involved in the control of expression, storage, translation and degradation of maternaltranscript essential to the oocyte and embryo development.

Controle molecular do acúmulo, tradução e degradação de mRNA em oócitos e embriões / Molecular control of accumulation, translation and degradation of mRNA in oocytes and embryos

A competência oocitária para suportar os posteriores estágios do desenvolvimento depende não somenteda correta segregação cromossômica e de transformações citoesqueléticas, mas, principalmente, da adequadatranscrição e estoque de mRNAs cruciais ao desenvolvimento e à viabilidade celular. Com a retomada dameiose, no entanto, embora o oócito mantenha a capacidade de tradução gênica e de síntese proteica, suaatividade transcricional é interrompida, sendo restabelecida apenas com a ativação do genoma embrionário.Deste modo, todo mRNA materno mobilizado durante maturação, expansão do cumulus, fertilização eembriogênese inicial deve ser sintetizado e estocado, em sua forma traducionalmente inativa, nos oócitosmantidos no estágio diplóteno da prófase I. Complexos mecanismos regulatórios, os quais envolvem apoliadenilação e a desadenilação do mRNA, estão implicados no processo de ativação e silenciamento tantotranscricional quanto traducional. Assim, dada à relevância do tema, esta revisão se propõe a abordar osprincipais eventos moleculares envolvidos no controle da expressão, do estoque, da tradução e da degradação detranscritos maternos imprescindíveis ao desenvolvimento oocitário e embrionário.(AU)

The oocyte competence to support the later stages of development depends not only on correctchromosome segregation and cytoskeletal changes, but mainly, the proper transcription and storage of mRNAscritical to the cellular development and viability. However, with the resumption of meiosis, although the oocytekept the ability of gene translation and protein synthesis, its transcriptional activity is interrupted and restoredonly with embryonic genome activation. Thus, all maternal mRNA mobilized during maturation, cumulusexpansion, fertilization and early embryogenesis must be synthesized and stored, in its translationally inactiveform, in oocytes kept in the diplotene stage of prophase I. Complex regulatory mechanisms which involvedeadenylation and polyadenylation of mRNA are involved in this process of activation and silencing astranscriptional as translational. So, due to the importance of the topic, this review proposes to discuss the mainmolecular events involved in the control of expression, storage, translation and degradation of maternaltranscript essential to the oocyte and embryo development.(AU)

Improving postcryopreservation survival capacity: an embryo-focused approach

The major challenge for a greater dissemination of in vitro produced (IVP) bovine embryos is to improve embryonic survival after cryoprese rvation. The involvement of embryo niclipids on this issue is well documented. However, it has been recognized that not only the amount of lipids that affects embryo cryotolerance, but the embryo survival capacity after cryopreservation is a rather multifactorial event. In this review, some strategies to improve embryonic lipid composition and postcryo preservation survival by modifying the embryos themselves to make them more cryopreservable are overviewed The ue of sedefined and defined serum free cul ture media, the addition of some chemicals in the culture media to modify embryo lipid composition, and the modulation of embryo cell membrane fluidity by cholesterol or unsaturated fatty acids added to the culture media and oocyte/embryo donor nutritional management with a diet enriched in polyunsaturated fatty acids, were described as alternatives for the improvement of IVP embryo survival after cryopreservation.(AU)

Improving postcryopreservation survival capacity: an embryo-focused approach

The major challenge for a greater dissemination of in vitro produced (IVP) bovine embryos is to improve embryonic survival after cryoprese rvation. The involvement of embryo niclipids on this issue is well documented. However, it has been recognized that not only the amount of lipids that affects embryo cryotolerance, but the embryo survival capacity after cryopreservation is a rather multifactorial event. In this review, some strategies to improve embryonic lipid composition and postcryo preservation survival by modifying the embryos themselves to make them more cryopreservable are overviewed The ue of sedefined and defined serum free cul ture media, the addition of some chemicals in the culture media to modify embryo lipid composition, and the modulation of embryo cell membrane fluidity by cholesterol or unsaturated fatty acids added to the culture media and oocyte/embryo donor nutritional management with a diet enriched in polyunsaturated fatty acids, were described as alternatives for the improvement of IVP embryo survival after cryopreservation.

Preservation of wild feline semen by freeze-drying: experimental model

According to the Convention on International Trade in Endangered Species, 36 wild feline species are threatened by extinction or severely endangered, and to save them is the target of several conservation programs. This study aimed to assess the viability of the freeze-drying technique for domestic cat sperm cells, with the ultimate goal of transferring this technology to the wild feline species. The do mestic cat is an excellent experimental model for wild felids. It is in this scenario that the freeze-drying process (low-temperature vacuum dehydration) of sperm cells shows its value in preserving male cats germplasm. Results from membrane and DNA integrity analysis are promising and validates the use of frozen-dried sperm samples in intracytoplasmic sperm injections (ICSIs). Further studies are still necessary to evaluate the ICSI embryo production using domestic cat frozen-dried sperm and the possibility of using such technology with wild felines.(AU)

Peculiaridades da coleta de oócitos para produção in vitro de embriões ovinos / Peculiarities of oocyte harvesting for in vitro production of sheep embryos

A expansão da ovinocultura brasileira impulsionou o desenvolvimento de novas biotécnicas de reprodução assistida visando ao aumento dos índices produtivos em concomitância com o acelerado progresso genético. Neste contexto, a produção in vitro de embriões (PIV) ovinos se intensificou, devido à possibilidade de otimização das fêmeas doadoras de oócitos e de incremento da quantidade de embriões produzidos. Entre os vários fatores implicados no sucesso dessa biotecnologia, a qualidade dos complexos cumulus-oophorus (COCs), caracterizada, principalmente, pela presença das células do cumulus, é considerada crucial. Deste modo, é imprescindível a ampliação do conhecimento referente aos diferentes métodos de coleta dos COCs e suas peculiaridades, visando a futuros aperfeiçoamentos biotecnológicos.(AU)

The expansion of Brazilian sheep rearing promoted the development of new biotechnologies of assisted reproduction in order to increase the production indices concomitantly with accelerated genetic progress. In this context, the in vitro production (IVP) of sheep embryos has intensified due to the possibility of optimization of the female oocytes donor and increase the number of embryos produced. Among the various factors involved in the success of this biotechnology, the cumulus-oophorus complexes (COCs) quality, characterized, mainly, by the presence of cumulus cells, is considered crucial. Thus, it is essential to increase the knowledge on the different methods of collection of COCs and their peculiarities, aiming for future biotechnological improvements. (AU)

Peculiaridades da coleta de oócitos para produção in vitro de embriões ovinos / Peculiarities of oocyte harvesting for in vitro production of sheep embryos

A expansão da ovinocultura brasileira impulsionou o desenvolvimento de novas biotécnicas de reprodução assistida visando ao aumento dos índices produtivos em concomitância com o acelerado progresso genético. Neste contexto, a produção in vitro de embriões (PIV) ovinos se intensificou, devido à possibilidade de otimização das fêmeas doadoras de oócitos e de incremento da quantidade de embriões produzidos. Entre os vários fatores implicados no sucesso dessa biotecnologia, a qualidade dos complexos cumulus-oophorus (COCs), caracterizada, principalmente, pela presença das células do cumulus, é considerada crucial. Deste modo, é imprescindível a ampliação do conhecimento referente aos diferentes métodos de coleta dos COCs e suas peculiaridades, visando a futuros aperfeiçoamentos biotecnológicos.

The expansion of Brazilian sheep rearing promoted the development of new biotechnologies of assisted reproduction in order to increase the production indices concomitantly with accelerated genetic progress. In this context, the in vitro production (IVP) of sheep embryos has intensified due to the possibility of optimization of the female oocytes donor and increase the number of embryos produced. Among the various factors involved in the success of this biotechnology, the cumulus-oophorus complexes (COCs) quality, characterized, mainly, by the presence of cumulus cells, is considered crucial. Thus, it is essential to increase the knowledge on the different methods of collection of COCs and their peculiarities, aiming for future biotechnological improvements.

Preservation of wild feline semen by freeze-drying: experimental model

According to the Convention on International Trade in Endangered Species, 36 wild feline species are threatened by extinction or severely endangered, and to save them is the target of several conservation programs. This study aimed to assess the viability of the freeze-drying technique for domestic cat sperm cells, with the ultimate goal of transferring this technology to the wild feline species. The do mestic cat is an excellent experimental model for wild felids. It is in this scenario that the freeze-drying process (low-temperature vacuum dehydration) of sperm cells shows its value in preserving male cats’ germplasm. Results from membrane and DNA integrity analysis are promising and validates the use of frozen-dried sperm samples in intracytoplasmic sperm injections (ICSIs). Further studies are still necessary to evaluate the ICSI embryo production using domestic cat frozen-dried sperm and the possibility of using such technology with wild felines.

Gargalos tecnológicos na reprodução assistida em ovinos: o estado da arte / Technological bottlenecks in sheep assisted reproduction: the state of the art

A expansão da criação de ovinos tem representado uma oportunidade no avanço dos estudos referentes às Biotécnicas de Reprodução Assistidas em ovinos. Como principal objetivo deste trabalho está uma análise dos principais gargalos tecnológicos à aplicação da Reprodução Assistida na espécie ovina. Serão ainda abordados princípios básicos que podem ser úteis à adoção de estratégias visando à superação desses entraves.

The expansion of the creation of sheep has represented an opportunity in the advancement of studies related to Biotechnology of Assisted Reproduction in sheep. The main objective of this study is an analysis of the major technological bottlenecks to the implementation of Assisted Reproduction in sheep. Basic principles that may be useful to adopt strategies aimed at overcoming these barriers will be approached.

Gargalos tecnológicos na reprodução assistida em ovinos: o estado da arte / Technological bottlenecks in sheep assisted reproduction: the state of the art

A expansão da criação de ovinos tem representado uma oportunidade no avanço dos estudos referentes às Biotécnicas de Reprodução Assistidas em ovinos. Como principal objetivo deste trabalho está uma análise dos principais gargalos tecnológicos à aplicação da Reprodução Assistida na espécie ovina. Serão ainda abordados princípios básicos que podem ser úteis à adoção de estratégias visando à superação desses entraves. (AU)

The expansion of the creation of sheep has represented an opportunity in the advancement of studies related to Biotechnology of Assisted Reproduction in sheep. The main objective of this study is an analysis of the major technological bottlenecks to the implementation of Assisted Reproduction in sheep. Basic principles that may be useful to adopt strategies aimed at overcoming these barriers will be approached. (AU)