Embryo competence and cryosurvival: Molecular and cellular features.

Global cattle genetic market is experiencing a change of strategy, large genetic companies, traditionally recognized in the artificial insemination field, have also begun to operate in the embryo market. Consequently, the demand for in vitro produced (IVP) embryos has grown. However, the overall efficiency of the biotechnology process remains low. Additionally, the lack of homogeneity of post-cryopreservation survival results of IVP embryos still impairing a massive dissemination of this biotechnology in the field. A great challenge for in vitro production labs is to increase the amount of embryos produced with exceptional quality after each round of in vitro fertilization. Herein, we discuss the molecular and cellular features associated with the competence and cryosurvival of IVP embryos. First, morphofunctional, cellular and molecular competence of the embryos were addressed and a relationship between embryo developmental ability and quality were established with cryosurvival and pregnancy success. Additionally, determinant factors of embryo competence and cryosurvival were discussed including the following effects: genotype, oocyte quality and follicular microenvironment, in vitro production conditions, and lipids and other determining molecules. Finally, embryo cryopreservation aspects were addressed and an embryo-focused approach to improve cryosurvival was presented.

Embryo competence and cryosurvival: Molecular and cellular features

Global cattle genetic market is experiencing a change of strategy, large genetic companies, traditionally recognized in the artificial insemination field, have also begun to operate in the embryo market. Consequently, the demand for in vitro produced (IVP) embryos has grown. However, the overall efficiency of the biotechnology process remains low. Additionally, the lack of homogeneity of post-cryopreservation survival results of IVP embryos still impairing a massive dissemination of this biotechnology in the field. A great challenge for in vitro production labs is to increase the amount of embryos produced with exceptional quality after each round of in vitro fertilization. Herein, we discuss the molecular and cellular features associated with the competence and cryosurvival of IVP embryos. First, morphofunctional, cellular and molecular competence of the embryos were addressed and a relationship between embryo developmental ability and quality were established with cryosurvival and pregnancy success. Additionally, determinant factors of embryo competence and cryosurvival were discussed including the following effects: genotype, oocyte quality and follicular microenvironment, in vitro production conditions, and lipids and other determining molecules. Finally, embryo cryopreservation aspects were addressed and an embryofocused approach to improve cryosurvival was presented.

Embryo competence and cryosurvival: Molecular and cellular features

Global cattle genetic market is experiencing a change of strategy, large genetic companies, traditionally recognized in the artificial insemination field, have also begun to operate in the embryo market. Consequently, the demand for in vitro produced (IVP) embryos has grown. However, the overall efficiency of the biotechnology process remains low. Additionally, the lack of homogeneity of post-cryopreservation survival results of IVP embryos still impairing a massive dissemination of this biotechnology in the field. A great challenge for in vitro production labs is to increase the amount of embryos produced with exceptional quality after each round of in vitro fertilization. Herein, we discuss the molecular and cellular features associated with the competence and cryosurvival of IVP embryos. First, morphofunctional, cellular and molecular competence of the embryos were addressed and a relationship between embryo developmental ability and quality were established with cryosurvival and pregnancy success. Additionally, determinant factors of embryo competence and cryosurvival were discussed including the following effects: genotype, oocyte quality and follicular microenvironment, in vitro production conditions, and lipids and other determining molecules. Finally, embryo cryopreservation aspects were addressed and an embryofocused approach to improve cryosurvival was presented.(AU)

Influence of commercially available follicle stimulating hormone on the in vitro maturation of bovine oocytes / Influência do hormônio folículo-estimulante disponível comercialmente sobre a maturação in vitro de oócitos bovinos

The work aimed (Experiment I) to compare commercial representations of porcine follicle stimulating hormone (FSH, Pluset® vs. Folltropin®) in concentration (10 μg/mL) and time (24 h) standard (more used in protocols of in vitro maturation, IVM); (Experiment II) to evaluate the best incubation time (6 h vs. 16 h vs. 24 h) and, (Experiment III) to analyze varying concentrations (1.0 μg/mL vs. 2.5 μg/mL vs. 10.0 μg/mL) of representations of FSH on the IVM of bovine oocytes. Thus, oocytes were recovered and submitted to IVM under appropriate conditions. After the IVM, oocytes were evaluated for expansion of cumulus cells (CCs), presence of the first polar body (1PB) and metaphase plate (MII). All the data were analyzed by the Fisher exact test (P 0.05) was observed in maturation rates of the oocytes incubated with FSH Pluset® or Folltropin®, assessed by expansion of CCs (97.6% vs. 94.3%), presence of 1PB (76.6% vs. 69.4%) and MII (70.0% vs. 68.6%). In Experiment II, when the incubation time with FSH was evaluated, both Pluset® as Folltropin® showed lower rate of expansion of CCs when they were present only in the first 6 h of IVM. As for presence of 1PB, differences were observed in relation to Pluset® while Folltropin® showed similar results in all incubation times. Regarding the MII, no difference was observed between the incubation times with FSH Pluset® and Folltropin®. In Experiment III, no difference was observed in the expansion of CCs, presence of 1PB and MII for concentrations evaluated FSH Pluset® and Folltropin®. Therefore, the FSH Pluset® and Folltropin® have the same efficiency in IVM of bovine oocytes. Regarding the incubation time, it is recommended to maintain FSH (Pluset® or Folltropin®) throughout the period of IVM, since there was no difference in the results of presence of MII...

O trabalho objetivou (Experimento I) comparar representações comerciais do hormônio folículo estimulante suíno (FSH, Pluset® vs. Folltropin®) na concentração (10 μg/mL) e tempo (24 h) padrão (mais utilizados nos protocolos de maturação in vitro, MIV); (Experimento II) avaliar o melhor tempo de incubação (6 h vs. 16 h vs. 24 h) e, (Experimento III) analisar concentrações variáveis (1,0 μg/mL vs. 2,5 μg/mL vs. 10,0 μg/mL) das representações de FSH sobre a MIV de oócitos bovinos. Para tanto, oócitos foram recuperados e submetidos a MIV em condições adequadas. Após a MIV, oócitos foram avaliados quanto à expansão das células do cumulus (CCs), presença do primeiro corpúsculo polar (1CP) e placa metafásica (MII). Todos os dados foram analisados pelo teste exato de Fisher (P 0,05) nas taxas de maturação de oócitos incubados com FSH Pluset® ou Folltropin®, avaliadas pela expansão das CCs (97,6% vs. 94,3%), presença de 1CP (76,6% vs. 69,4%) e MII (70,0% vs. 68,6%). No Experimento II, quanto ao tempo de incubação com FSH foi avaliado, tanto Pluset® quanto Folltropin® mostraram uma menor taxa de expansão de CCs somente nas primeiras 6 h de MIV. Quanto à presença de 1CP, diferenças foram observadas em relação ao Pluset® enquanto o Folltropin® mostrou resultados similares em todos os tempos de incubação. Em relação à MII, nenhuma diferença foi observada entre os tempos de incubação com o FSH Pluset® e Folltropin®. No Experimento III, nenhuma diferença foi observada na expansão das CCs, presença do 1CP e MII para as concentrações avaliadas de FSH Pluset® e Folltropin®. Portanto, FSH Pluset® e Folltropin® tem a mesma eficiência na MIV de oócitos bovinos. Em relação ao tempo de incubação, é recomendado manter o FSH (Pluset® or Folltropin®) ao longo do período da MIV, uma vez que nenhuma diferença foi observada nos resultados da presença de MII...

Influence of commercially available follicle stimulating hormone on the in vitro maturation of bovine oocytes / Influência do hormônio folículo-estimulante disponível comercialmente sobre a maturação in vitro de oócitos bovinos

The work aimed (Experiment I) to compare commercial representations of porcine follicle stimulating hormone (FSH, Pluset® vs. Folltropin®) in concentration (10 μg/mL) and time (24 h) standard (more used in protocols of in vitro maturation, IVM); (Experiment II) to evaluate the best incubation time (6 h vs. 16 h vs. 24 h) and, (Experiment III) to analyze varying concentrations (1.0 μg/mL vs. 2.5 μg/mL vs. 10.0 μg/mL) of representations of FSH on the IVM of bovine oocytes. Thus, oocytes were recovered and submitted to IVM under appropriate conditions. After the IVM, oocytes were evaluated for expansion of cumulus cells (CCs), presence of the first polar body (1PB) and metaphase plate (MII). All the data were analyzed by the Fisher exact test (P < 0.05). Initially, in Experiment I, no difference (P > 0.05) was observed in maturation rates of the oocytes incubated with FSH Pluset® or Folltropin®, assessed by expansion of CCs (97.6% vs. 94.3%), presence of 1PB (76.6% vs. 69.4%) and MII (70.0% vs. 68.6%). In Experiment II, when the incubation time with FSH was evaluated, both Pluset® as Folltropin® showed lower rate of expansion of CCs when they were present only in the first 6 h of IVM. As for presence of 1PB, differences were observed in relation to Pluset® while Folltropin® showed similar results in all incubation times. Regarding the MII, no difference was observed between the incubation times with FSH Pluset® and Folltropin®. In Experiment III, no difference was observed in the expansion of CCs, presence of 1PB and MII for concentrations evaluated FSH Pluset® and Folltropin®. Therefore, the FSH Pluset® and Folltropin® have the same efficiency in IVM of bovine oocytes. Regarding the incubation time, it is recommended to maintain FSH (Pluset® or Folltropin®) throughout the period of IVM, since there was no difference in the results of presence of MII...(AU)

O trabalho objetivou (Experimento I) comparar representações comerciais do hormônio folículo estimulante suíno (FSH, Pluset® vs. Folltropin®) na concentração (10 μg/mL) e tempo (24 h) padrão (mais utilizados nos protocolos de maturação in vitro, MIV); (Experimento II) avaliar o melhor tempo de incubação (6 h vs. 16 h vs. 24 h) e, (Experimento III) analisar concentrações variáveis (1,0 μg/mL vs. 2,5 μg/mL vs. 10,0 μg/mL) das representações de FSH sobre a MIV de oócitos bovinos. Para tanto, oócitos foram recuperados e submetidos a MIV em condições adequadas. Após a MIV, oócitos foram avaliados quanto à expansão das células do cumulus (CCs), presença do primeiro corpúsculo polar (1CP) e placa metafásica (MII). Todos os dados foram analisados pelo teste exato de Fisher (P < 0,05). Inicialmente, no Experimento I, nenhuma diferença foi observada (P > 0,05) nas taxas de maturação de oócitos incubados com FSH Pluset® ou Folltropin®, avaliadas pela expansão das CCs (97,6% vs. 94,3%), presença de 1CP (76,6% vs. 69,4%) e MII (70,0% vs. 68,6%). No Experimento II, quanto ao tempo de incubação com FSH foi avaliado, tanto Pluset® quanto Folltropin® mostraram uma menor taxa de expansão de CCs somente nas primeiras 6 h de MIV. Quanto à presença de 1CP, diferenças foram observadas em relação ao Pluset® enquanto o Folltropin® mostrou resultados similares em todos os tempos de incubação. Em relação à MII, nenhuma diferença foi observada entre os tempos de incubação com o FSH Pluset® e Folltropin®. No Experimento III, nenhuma diferença foi observada na expansão das CCs, presença do 1CP e MII para as concentrações avaliadas de FSH Pluset® e Folltropin®. Portanto, FSH Pluset® e Folltropin® tem a mesma eficiência na MIV de oócitos bovinos. Em relação ao tempo de incubação, é recomendado manter o FSH (Pluset® or Folltropin®) ao longo do período da MIV, uma vez que nenhuma diferença foi observada nos resultados da presença de MII...(AU)

Adição da proteína específica do oviduto de porcas (pOSP) e da melatonina em meios de maturação e o efeito na clivagem in vitro de embriões suínos / Addition of the oviduct specific protein of porcine (pOSP) and melatonin in maturation media and their effect on the in vitro production of pig embryos

No presente estudo, utilizou-se a melatonina e a proteína específica do oviduto (pOSP) nos meios de maturação in vitro. Foram avaliadas a expansão do complexo cumulus-ovócito (CCOs), as concentrações intracelulares de espécies reativas de oxigênio (ROS) e o desenvolvimento embrionário nos diferentes grupos (C = controle; T1 = somente com melatonina; T2 = com melatonina e pOSP e T3 somente com pOSP). No tocante à expansão do CCOs, houve diferença (P<0,05) dos valores obtidos no grupo C em relação aos valores médios dos grupos T1, T2 e T3, porém não houve diferença entre os valores obtidos nos tratamentos (P>0,05). Na dosagem de ROS, não houve diferença entre os valores médios obtidos no grupo C (26,4±10,9) e o valor verificado no grupo T1 (23,4±7,8), porém no grupo T2 (21,3±9,7) o valor médio mostrou-se satisfatório em relação ao valor do grupo C. No entanto, o valor médio do grupo T3 (16,6±10,5) foi o que demonstrou resultado mais satisfatório quando comparado aos demais grupos (P<0,05). A produção de embriões foi avaliada por meio da taxa de clivagem. Não houve diferença (P >0,05) entre os valores obtidos entre o grupo C (48,9 %) e os valores verificados nos grupos T1 (51,5 %), T2 (50 %), T3 (57,7 %), nem destes entre si. Este estudo permitiu concluir que a proteína específica do oviduto recombinante e a melatonina foram eficientes em melhorar a expansão dos CCOs. Além disso, as células tratadas com pOSP mostraram-se com menor quantidade de ROS, podendo a pOSP ser considerada um antioxidante proteico.(AU)

The present study used melatonin and recombinant oviduct specific protein (pOSP) in in vitro maturation medium (IVM). The expansion of the cumulus-oocyte complexes (COCs), the intracellular concentrations of reactive oxygen species (ROS) and embryo development of the different groups were evaluated (C = control; T1 = melatonin; T2 = melatonin and pOSP and T3 = pOSP). Regarding the COCs expansion, the groups T1, T2 and T3 showed satisfactory results compared with group C (P<0.05), but there was no difference between treatments (P>0.05). In the ROS dosage, there was no difference between the mean values obtained in group C (26.4 ± 10.9) and group 1 (23.4 ± 7.8). However, in group 2 (21.3 ± 9.7), the average value was found to be satisfactory in relation group C. Despite that, the average value of treatment 3 (16.6 ± 10.5) was the most satisfactory result found compared to the other groups (P<0.05). The production of embryos was evaluated by cleavage rate, there was no difference between the values obtained in group C and the values recorded in groups T1 (51.5 %), T2 (50 %), T3 (57.7 %), and among them. This study showed that the pOSP and the melatonin were effective in the improvement of the expansion of COCs cells. In addition, the cells that were treated with pOSP presented a lower amount of ROS, allowing the pOSP to be considered a proteic antioxidant.(AU)

Adição da proteína específica do oviduto de porcas (pOSP) e da melatonina em meios de maturação e o efeito na clivagem in vitro de embriões suínos / Addition of the oviduct specific protein of porcine (pOSP) and melatonin in maturation media and their effect on the in vitro production of pig embryos

No presente estudo, utilizou-se a melatonina e a proteína específica do oviduto (pOSP) nos meios de maturação in vitro. Foram avaliadas a expansão do complexo cumulus-ovócito (CCOs), as concentrações intracelulares de espécies reativas de oxigênio (ROS) e o desenvolvimento embrionário nos diferentes grupos (C = controle; T1 = somente com melatonina; T2 = com melatonina e pOSP e T3 somente com pOSP). No tocante à expansão do CCOs, houve diferença (P<0,05) dos valores obtidos no grupo C em relação aos valores médios dos grupos T1, T2 e T3, porém não houve diferença entre os valores obtidos nos tratamentos (P>0,05). Na dosagem de ROS, não houve diferença entre os valores médios obtidos no grupo C (26,4±10,9) e o valor verificado no grupo T1 (23,4±7,8), porém no grupo T2 (21,3±9,7) o valor médio mostrou-se satisfatório em relação ao valor do grupo C. No entanto, o valor médio do grupo T3 (16,6±10,5) foi o que demonstrou resultado mais satisfatório quando comparado aos demais grupos (P<0,05). A produção de embriões foi avaliada por meio da taxa de clivagem. Não houve diferença (P >0,05) entre os valores obtidos entre o grupo C (48,9 %) e os valores verificados nos grupos T1 (51,5 %), T2 (50 %), T3 (57,7 %), nem destes entre si. Este estudo permitiu concluir que a proteína específica do oviduto recombinante e a melatonina foram eficientes em melhorar a expansão dos CCOs. Além disso, as células tratadas com pOSP mostraram-se com menor quantidade de ROS, podendo a pOSP ser considerada um antioxidante proteico.(AU)

The present study used melatonin and recombinant oviduct specific protein (pOSP) in in vitro maturation medium (IVM). The expansion of the cumulus-oocyte complexes (COCs), the intracellular concentrations of reactive oxygen species (ROS) and embryo development of the different groups were evaluated (C = control; T1 = melatonin; T2 = melatonin and pOSP and T3 = pOSP). Regarding the COCs expansion, the groups T1, T2 and T3 showed satisfactory results compared with group C (P<0.05), but there was no difference between treatments (P>0.05). In the ROS dosage, there was no difference between the mean values obtained in group C (26.4 ± 10.9) and group 1 (23.4 ± 7.8). However, in group 2 (21.3 ± 9.7), the average value was found to be satisfactory in relation group C. Despite that, the average value of treatment 3 (16.6 ± 10.5) was the most satisfactory result found compared to the other groups (P<0.05). The production of embryos was evaluated by cleavage rate, there was no difference between the values obtained in group C and the values recorded in groups T1 (51.5 %), T2 (50 %), T3 (57.7 %), and among them. This study showed that the pOSP and the melatonin were effective in the improvement of the expansion of COCs cells. In addition, the cells that were treated with pOSP presented a lower amount of ROS, allowing the pOSP to be considered a proteic antioxidant.(AU)

Vantagens e desafios das biotécnicas avançadas utilizadas na reprodução equina assistida / Advantages and challenges of advanced biotech used in assisted equine reproduction

Relatos apontam que a espécie equina foi a primeira a ser submetida à inseminação artificial. Apesar deste fato, o desenvolvimento da reprodução assistida nos equinos foi lento em comparação às demais espécies voltadas diretamente para a produção animal. A liberação do uso da transferência de embriões pela maioria das associações de raças estimulou o desenvolvimento e a expansão de algumas técnicas de multiplicação animal para uso em equinos. A presente revisão teve por objetivo abordar as biotécnicas avançadas atualmente disponíveis para a espécie, como a superovulação, a transferência de ovócitos, a transferência intratubárica de gametas, a injeção intracitoplasmática de gametas, a produção in vitro de embriões e a clonagem, enfatizando suas aplicações prática e clínica, bem como determinados desafios que ainda precisam ser superados em cada biotécnica.(AU)

Some reports suggest the equine species as the first one to be subjected to the artificial insemination. Despite this fact, the development of assisted reproduction in this species has been slow when compared to other production species. The authorization of the use of embryo transfer by some breed associations stimulated the development and expansion of several reproductive techniques for use in horses. The present study aimed to address the advanced biotechnologies currently available for this species, such as superovulation, transfer of oocytes, gamete intrafallopian transfer, in vitro production of embryos and cloning, emphasizing their practical and clinical applications, as well as certain challenges that still need to be overcome in each biotech.(AU)

Vantagens e desafios das biotécnicas avançadas utilizadas na reprodução equina assistida / Advantages and challenges of advanced biotech used in assisted equine reproduction

Relatos apontam que a espécie equina foi a primeira a ser submetida à inseminação artificial. Apesar deste fato, o desenvolvimento da reprodução assistida nos equinos foi lento em comparação às demais espécies voltadas diretamente para a produção animal. A liberação do uso da transferência de embriões pela maioria das associações de raças estimulou o desenvolvimento e a expansão de algumas técnicas de multiplicação animal para uso em equinos. A presente revisão teve por objetivo abordar as biotécnicas avançadas atualmente disponíveis para a espécie, como a superovulação, a transferência de ovócitos, a transferência intratubárica de gametas, a injeção intracitoplasmática de gametas, a produção in vitro de embriões e a clonagem, enfatizando suas aplicações prática e clínica, bem como determinados desafios que ainda precisam ser superados em cada biotécnica.

Some reports suggest the equine species as the first one to be subjected to the artificial insemination. Despite this fact, the development of assisted reproduction in this species has been slow when compared to other production species. The authorization of the use of embryo transfer by some breed associations stimulated the development and expansion of several reproductive techniques for use in horses. The present study aimed to address the advanced biotechnologies currently available for this species, such as superovulation, transfer of oocytes, gamete intrafallopian transfer, in vitro production of embryos and cloning, emphasizing their practical and clinical applications, as well as certain challenges that still need to be overcome in each biotech.