Effects of different stimulators of cGMP synthesis on lipid content in bovine oocytes matured in vitro

Abstract Bovine oocytes and blastocysts produced in vitro are frequently of lower quality and less cryotolerant than those produced in vivo, and greater accumulation of lipids in the cytoplasm has been pointed out as one of the reasons. In human adipocytes cGMP signaling through the activation of PKG appears to be involved in lipid metabolism, and components of this pathway have been detected in bovine cumulus-oocyte complexes (COCs). The aim of this study was to investigate the influence of this pathway on the lipid content in oocytes and expression of PLIN2 (a lipid metabolism-related gene) in cumulus cells. COCs were matured in vitro for 24 h with different stimulators of cGMP synthesis. The activation of soluble guanylyl cyclase (sGC) by Protoporphyrin IX reduced lipid content (22.7 FI) compared to control oocytes (36.45 FI; P <0.05). Stimulation of membrane guanylyl cyclase (mGC) with natriuretic peptides precursors A and C (NPPA and NPPC) had no effect (36.5 FI; P>0.05). When the PKG inhibitor KT5823 was associated with Protoporphyrin IX, its effect was reversed and lipid contents increased (52.71 FI; P<0.05). None of the stimulators of cGMP synthesis affected the expression of PLIN2 in cumulus cells. In conclusion, stimulation of sGC for cGMP synthesis promotes lipolytic activities in bovine oocytes matured in vitro and such effect is mediated by PKG. However, such effect may vary depending on the stimulus received and/or which synthesis enzyme was activated, as stimulation of mGC had no effects.

Effects of different stimulators of cGMP synthesis on lipid content in bovine oocytes matured in vitro

Bovine oocytes and blastocysts produced in vitro are frequently of lower quality and less cryotolerant than those produced in vivo, and greater accumulation of lipids in the cytoplasm has been pointed out as one of the reasons. In human adipocytes cGMP signaling through the activation of PKG appears to be involved in lipid metabolism, and components of this pathway have been detected in bovine cumulus-oocyte complexes (COCs). The aim of this study was to investigate the influence of this pathway on the lipid content in oocytes and expression of PLIN2 (a lipid metabolism-related gene) in cumulus cells. COCs were matured in vitro for 24 h with different stimulators of cGMP synthesis. The activation of soluble guanylyl cyclase (sGC) by Protoporphyrin IX reduced lipid content (22.7 FI) compared to control oocytes (36.45 FI; P <0.05). Stimulation of membrane guanylyl cyclase (mGC) with natriuretic peptides precursors A and C (NPPA and NPPC) had no effect (36.5 FI; P>0.05). When the PKG inhibitor KT5823 was associated with Protoporphyrin IX, its effect was reversed and lipid contents increased (52.71 FI; P<0.05). None of the stimulators of cGMP synthesis affected the expression of PLIN2 in cumulus cells. In conclusion, stimulation of sGC for cGMP synthesis promotes lipolytic activities in bovine oocytes matured in vitro and such effect is mediated by PKG. However, such effect may vary depending on the stimulus received and/or which synthesis enzyme was activated, as stimulation of mGC had no effects.(AU)

In vitro embryos production of oocytes selected based on follicular size and by brilliant cresyl blue staining / Produção in vitro de embriões de oócitos selecionados baseados no tamanho folicular e por coloração com azul cresil brilhante

We evaluated the competence of bovine oocytes via morphological selection associated with follicular diameter and by brilliant cresyl blue (BCB) staining, in order to improve oocyte selection and the efficiency of in vitro embryo production. Follicles with small (2<4 mm), large (4–8 mm) or mixed (2–8 mm) diameters were aspirated from slaughterhouse ovaries. Next, the morphologically categorized oocytes were stained and evaluated with 26 μM of BCB (90 min), followed by an assessment of in vitro maturation (MII) and blastocyst production. Oocytes from the large (61.1 ± 3.9%) and mixed (52.9 ± 4.6%) follicular groups showed comparable (p>0,05) BCB+ rates, but both differed (p<0.05) as compared to the small-diameter group (41.0 ± 4.5%). Between the BCB+ and BCB oocytes, there was no difference (p>0,05) in MII rate in the mixed (respectively 86.7 ± 3.4 and 75.8 ± 2.2), large (respectively 89.4 ± 2.6 and 79.7 ± 2.3) and small (respectively 76.7 ± 2.9 and 67.2 ± 2.5) diameter groups. Comparing BCB (+ and -) oocytes, we observed (p>0,05) no change in the percentage of embryos in the large group (respectively 67.3 ± 3.3 and 53.5 ± 5.8) and in the small group (respectively 43.8 ± 5.3 and 33.9 ± 2.2), but there was a difference (p<0.05) in the mixed group (respectively 57.8 ± 3.9 and 39.6 ± 7.5). Furthermore, when we compared between follicular groups, the BCB+/large-diameter group (61.1 ± 3.9%) had a significant higher percentage of embryos (p<0.05) than the small-diameter BCB+ (43.8 ± 5.3) groups. To conclude, selection procedures that use follicle size and/or BCB staining may be used as an additional tool to improve the in vitro production of embryos.(AU)

Avaliou-se a competência de oócitos bovinos através da seleção morfológica associada ao diâmetro folicular e pela coloração azul cresil brilhante (BCB), a fim de melhorar a seleção de oócitos e a eficiência da produção in vitro de embriões. Folículos com diâmetro pequeno (2<4 mm), grande (4-8 mm) ou misto (2-8 mm) foram aspirados de ovários de matadouro. Em seguida, os oócitos classificados morfologicamente foram corados e avaliados com 26 μM de BCB (90 min), seguido de uma avaliação da maturação in vitro (MII) e produção de blastocisto. Os oócitos dos grupos folículo grande (61,1 ± 3,9%) e misto (52,9 ± 4,6%) apresentaram taxas de BCB+ comparáveis (p>0,05), mas ambos diferiram (p<0,05) em relação ao grupo folículo pequeno (41,0 ± 4,5%). Entre os oócitos BCB+ e BCB–, não houve diferença (p>0,05) na taxa de MII nos grupos mistos (respectivamente 86,7 ± 3,4 e 75,8 ± 2,2), grande (respectivamente 89,4 ± 2,6 e 79,7 ± 2,3) e pequeno (respectivamente 76,7 ± 2,9 e 67,2 ± 2,5). Comparando os oócitos BCB (+ e -), não observamos alteração (p>0,05) na porcentagem de embriões no grupo grande (respectivamente 67,3 ± 3,3 e 53,5 ± 5,8) e no grupo pequeno (respectivamente 43,8 ± 5,3 e 33,9 ± 2,2), mas há houve diferença (p<0,05) no grupo misto (respectivamente 57,8 ± 3,9 e 39,6 ± 7,5). Além disso, quando comparamos os grupos foliculares, o grupo BCB+/grande (61,1 ± 3,9%) apresentou uma porcentagem significativamente maior de embriões (p<0,05) do que o grupo BCB+/pequeno (43,8 ± 5,3). Para concluir, os procedimentos de seleção que usam tamanho do folículo e/ou coloração com BCB podem ser usados como uma ferramenta adicional para melhorar a produção in vitro de embriões.(AU)

In vitro embryos production of oocytes selected based on follicular size and by brilliant cresyl blue staining / Produção in vitro de embriões de oócitos selecionados baseados no tamanho folicular e por coloração com azul cresil brilhante

We evaluated the competence of bovine oocytes via morphological selection associated with follicular diameter and by brilliant cresyl blue (BCB) staining, in order to improve oocyte selection and the efficiency of in vitro embryo production. Follicles with small (20,05) BCB+ rates, but both differed (p0,05) in MII rate in the mixed (respectively 86.7 ± 3.4 and 75.8 ± 2.2), large (respectively 89.4 ± 2.6 and 79.7 ± 2.3) and small (respectively 76.7 ± 2.9 and 67.2 ± 2.5) diameter groups. Comparing BCB (+ and -) oocytes, we observed (p>0,05) no change in the percentage of embryos in the large group (respectively 67.3 ± 3.3 and 53.5 ± 5.8) and in the small group (respectively 43.8 ± 5.3 and 33.9 ± 2.2), but there was a difference (p<0.05) in the mixed group (respectively 57.8 ± 3.9 and 39.6 ± 7.5). Furthermore, when we compared between follicular groups, the BCB+/large-diameter group (61.1 ± 3.9%) had a significant higher percentage of embryos (p<0.05) than the small-diameter BCB+ (43.8 ± 5.3) groups. To conclude, selection procedures that use follicle size and/or BCB staining may be used as an additional tool to improve the in vitro production of embryos.

Avaliou-se a competência de oócitos bovinos através da seleção morfológica associada ao diâmetro folicular e pela coloração azul cresil brilhante (BCB), a fim de melhorar a seleção de oócitos e a eficiência da produção in vitro de embriões. Folículos com diâmetro pequeno (20,05), mas ambos diferiram (p0,05) na taxa de MII nos grupos mistos (respectivamente 86,7 ± 3,4 e 75,8 ± 2,2), grande (respectivamente 89,4 ± 2,6 e 79,7 ± 2,3) e pequeno (respectivamente 76,7 ± 2,9 e 67,2 ± 2,5). Comparando os oócitos BCB (+ e -), não observamos alteração (p>0,05) na porcentagem de embriões no grupo grande (respectivamente 67,3 ± 3,3 e 53,5 ± 5,8) e no grupo pequeno (respectivamente 43,8 ± 5,3 e 33,9 ± 2,2), mas há houve diferença (p<0,05) no grupo misto (respectivamente 57,8 ± 3,9 e 39,6 ± 7,5). Além disso, quando comparamos os grupos foliculares, o grupo BCB+/grande (61,1 ± 3,9%) apresentou uma porcentagem significativamente maior de embriões (p<0,05) do que o grupo BCB+/pequeno (43,8 ± 5,3). Para concluir, os procedimentos de seleção que usam tamanho do folículo e/ou coloração com BCB podem ser usados como uma ferramenta adicional para melhorar a produção in vitro de embriões.

Sistemas de manejo para maximização da eficiência reprodutiva em bovinos de corte nos trópicos / Management systems to maximize the reproductive efficiency in beef cattle in the tropics / Sistemas de gestión para maximizar la eficiencia reproductiva en ganado de carne em las zonas tropicales

Na bovinocultura de corte, a reprodução pode ser considerada o principal pilar de sustentação da cadeia, visto que produz a matéria-prima elementar dessa indústria: o bezerro, determinando diretamente a eficiência da produção animal e garantindo o sucesso dos sistemas de produção. Desta forma, a alta eficiência reprodutiva do rebanho, atrelada a elevados índices de produção, devem ser metas consolidadas dentre os criadores. O Brasil é um país com grande potencial para produção de carne bovina, no entanto, ainda apresenta baixa eficiência produtiva e reprodutiva, com índices reprodutivos muito baixos e insatisfatórios, além de um manejo muitas vezes precário, com pouco ou nenhum controle acerca das diferentes fases que envolvem o ano reprodutivo, como seleção dos reprodutores, estação de monta, estação de parição e processo de desmama. Neste contexto, o planejamento reprodutivo em uma propriedade e a inclusão de estratégias de manejo que maximizem a eficiência reprodutiva, torna-se crucial e de suma importância para a melhoria do desempenho produtivo e, consequentemente, da lucratividade na criação de rebanhos comerciais. A adoção de um adequado manejo reprodutivo, assim como sanitário e nutricional, também está intimamente atrelada à máxima exploração do potencial genético dos animais. Diante da grande importância da reprodução na cadeia produtiva da carne bovina, esta revisão visou reunir e apresentar as principais e mais eficientes estratégias de manejo adotadas para maximização do desempenho reprodutivo e produtivo em rebanhos de corte.(AU)

In beef cattle, the reproduction can be considered the main pillar of the chain support, since it produces the basic raw material of the industry: the calf, directly determining the efficiency of animal production and ensuring the success of the production systems. Thus, high reproductive efficiency of the herd, linked at high production rates must be consolidated targets among creators. Brazil is a country with great potential for beef production, however, still has a low productive and reproductive efficiency, with very low and unsatisfactory reproductive rates, plus a handling often precarious, with little or no control on the different phases involving the reproductive year, as selection of breeding, breeding season, calving season and weaning process. In this context, the reproductive planning on a property and the inclusion of management strategies that maximize reproductive efficiency, it is crucial and very important for improving growth performance and hence profitability in the creation of commercial herds. The adoption of an adequate reproductive management, as well as health and nutrition, is also closely linked to the maximum exploitation of the genetic potential of the animals. Due the great importance of reproduction in the production chain of beef, this review aimed to gather and present the main and most effective management strategies adopted to maximize the reproductive and productive performance in beef cattle herds.(AU)

En la producción de ganado de carne, la reproducción puede ser considerado como el pilar principal del soporte de la cadena, ya que produce la materia prima básica de la industria: el becerro, determinando directamente la eficiencia de la producción animal y garantizar el éxito de los sistemas de producción. Por lo tanto, una alta eficiencia reproductiva del rebaño, vinculado a altas tasas de producción debe consolidarse objetivos entre los creadores. Brasil es un país con un gran potencial para la producción de carne, sin embargo, todavía tiene una eficiencia productiva y reproductiva baja, con tasas de reproducción muy baja y poco satisfactorios, además de un manejo a menudo precaria, con poco o ningún control sobre las diferentes fases que implican el año reproductiva, como la selección de crianza, estación de cría, época de parto y el proceso de destete. En este contexto, la planificación reproductiva en una propiedad y la inclusión de estrategias de gestión que maximicen la eficiencia reproductiva, es crucial y muy importante para mejorar el rendimiento del crecimiento y por lo tanto la rentabilidad en la creación de los rebaños comerciales. La adopción de un manejo reproductivo adecuado, así como la salud y la nutrición, está también estrechamente vinculados a la explotación máxima del potencial genético de los animales. Debido a la gran importancia de la reproducción en la cadena de producción de carne de vacuno, esta revisión tuvo como objetivo reunir y presentar los principales y más efectivas estrategias de gestión adoptadas para maximizar el rendimiento reproductivo y productivo en los rebaños de ganado vacuno.(AU)

Sistemas de manejo para maximização da eficiência reprodutiva em bovinos de corte nos trópicos / Management systems to maximize the reproductive efficiency in beef cattle in the tropics / Sistemas de gestión para maximizar la eficiencia reproductiva en ganado de carne em las zonas tropicales

Na bovinocultura de corte, a reprodução pode ser considerada o principal pilar de sustentação da cadeia, visto que produz a matéria-prima elementar dessa indústria: o bezerro, determinando diretamente a eficiência da produção animal e garantindo o sucesso dos sistemas de produção. Desta forma, a alta eficiência reprodutiva do rebanho, atrelada a elevados índices de produção, devem ser metas consolidadas dentre os criadores. O Brasil é um país com grande potencial para produção de carne bovina, no entanto, ainda apresenta baixa eficiência produtiva e reprodutiva, com índices reprodutivos muito baixos e insatisfatórios, além de um manejo muitas vezes precário, com pouco ou nenhum controle acerca das diferentes fases que envolvem o ano reprodutivo, como seleção dos reprodutores, estação de monta, estação de parição e processo de desmama. Neste contexto, o planejamento reprodutivo em uma propriedade e a inclusão de estratégias de manejo que maximizem a eficiência reprodutiva, torna-se crucial e de suma importância para a melhoria do desempenho produtivo e, consequentemente, da lucratividade na criação de rebanhos comerciais. A adoção de um adequado manejo reprodutivo, assim como sanitário e nutricional, também está intimamente atrelada à máxima exploração do potencial genético dos animais. Diante da grande importância da reprodução na cadeia produtiva da carne bovina, esta revisão visou reunir e apresentar as principais e mais eficientes estratégias de manejo adotadas para maximização do desempenho reprodutivo e produtivo em rebanhos de corte.

In beef cattle, the reproduction can be considered the main pillar of the chain support, since it produces the basic raw material of the industry: the calf, directly determining the efficiency of animal production and ensuring the success of the production systems. Thus, high reproductive efficiency of the herd, linked at high production rates must be consolidated targets among creators. Brazil is a country with great potential for beef production, however, still has a low productive and reproductive efficiency, with very low and unsatisfactory reproductive rates, plus a handling often precarious, with little or no control on the different phases involving the reproductive year, as selection of breeding, breeding season, calving season and weaning process. In this context, the reproductive planning on a property and the inclusion of management strategies that maximize reproductive efficiency, it is crucial and very important for improving growth performance and hence profitability in the creation of commercial herds. The adoption of an adequate reproductive management, as well as health and nutrition, is also closely linked to the maximum exploitation of the genetic potential of the animals. Due the great importance of reproduction in the production chain of beef, this review aimed to gather and present the main and most effective management strategies adopted to maximize the reproductive and productive performance in beef cattle herds.

En la producción de ganado de carne, la reproducción puede ser considerado como el pilar principal del soporte de la cadena, ya que produce la materia prima básica de la industria: el becerro, determinando directamente la eficiencia de la producción animal y garantizar el éxito de los sistemas de producción. Por lo tanto, una alta eficiencia reproductiva del rebaño, vinculado a altas tasas de producción debe consolidarse objetivos entre los creadores. Brasil es un país con un gran potencial para la producción de carne, sin embargo, todavía tiene una eficiencia productiva y reproductiva baja, con tasas de reproducción muy baja y poco satisfactorios, además de un manejo a menudo precaria, con poco o ningún control sobre las diferentes fases que implican el año reproductiva, como la selección de crianza, estación de cría, época de parto y el proceso de destete. En este contexto, la planificación reproductiva en una propiedad y la inclusión de estrategias de gestión que maximicen la eficiencia reproductiva, es crucial y muy importante para mejorar el rendimiento del crecimiento y por lo tanto la rentabilidad en la creación de los rebaños comerciales. La adopción de un manejo reproductivo adecuado, así como la salud y la nutrición, está también estrechamente vinculados a la explotación máxima del potencial genético de los animales. Debido a la gran importancia de la reproducción en la cadena de producción de carne de vacuno, esta revisión tuvo como objetivo reunir y presentar los principales y más efectivas estrategias de gestión adoptadas para maximizar el rendimiento reproductivo y productivo en los rebaños de ganado vacuno.

Proteomics, metabolomics and lipidomics in reproductive biotechnologies: the MS solutions

Background : A broader view of living systems complexity is bringing important contributions to biological sciences, since the genome expression is affected by other classes of molecules, which in their turn interact themselves in cellular metabolic pathways and biochemical networks. This level of information has been made possible by the emergence of the omic strategies, such as proteomics, metabolomics and lipidomics, that are mainly based on mass spectrometry (MS) platforms. MS has presented an incredible development over the last years, evolving to a powerful and universal analytical technique. Its ability to analyze proteins and small molecules such as lipids, sugars and metabolites at the structural level, with sensitivity and speed inconceivable a few years ago, is the major driving force in the omic fields. The development of electrospray and matrix-assisted laser desorption/ionization (MALDI) ionization techniques has decisively contributed to the many applications of this technology nowadays. Herein, we present and discuss omic concepts and strategies, as well as detail basic principles of MS. Applications and future perspectives of these approaches are focused in the reproductive medicine area. Review: The omic technologies propose global characterization of specific classes of target biomolecules of cellular systems as a strategy to achieve comprehensive understanding of biological functions. The genomics, aimed at performing the entire genetic sequencing of organisms, represented the seminal step towards the understanding of the complex logic that orchestrates the function of all organisms or the defects leading to diseases. But to express the phenotype, information needs to flow from DNA via carrier biomolecules through processes that are being addressed by new omic sciences such as the transcriptomics, proteomics, metabolomics, glycomics, lipidomics, and fluxomics. Mass spectrometry (MS) is nowadays the most powerful technique for the structural characterization of biomolecules, and has therefore become the central technique for the omic sciences. Using revolutionary ionization techniques such as electrospray (ESI) and matrix-assisted laser desorption ionization (MALDI), a wide range of biomolecules such as peptides, proteins, lipids and sugars are efficiently transferred in intact ionized forms to the gas phase for MS analysis. The development of ESI-MS and MALDI-MS has been awarded the Nobel Prize for Chemistry in 2002, rocketing the application of MS in the omic sciences. More recently, ambient ionization MS techniques, such as desorption electrospray ionization (DESI) and easy ambient sonic-spray ionization (EASI), have been developed for ionization in the open atmosphere, in a workup free and high throughput fashion directly from sample in their original environments. For the more complex samples, the coupling with separation techniques such as liquid chromatrography (LC) as well as the use of tandem MS (LC-MS/MS) has allowed comprehensive mixture characterization of major biomolecules. Conclusion: This manuscript describes recent advances of MS in the proteomics, metabolomics and lipidomics for biological sciences, and points out the relevant contributions that MS is likely to bring to fundamental and applied research in human and animal embryo biotechnologies.

Bissecação, congelação e sobrevivencia in vivo de embriões murinos

pt