Evolução da utilização de espermatozoides sexados / Evolution of sexed spermatozoa utilization

A sexagem de espermatozoides tem relevante aplicação na produção animal. Várias publicações demonstram a eficiência do processo de sexagem por citometria de fluxo que separa os espermatozoides X e Y em função da mensuração do conteúdo de DNA. Importantes modificações no processo de sexagem por citometria de fluxo, tais como pressão, intensidade do laser, diluidores tem levado à diminuição dos danos nos espermatozoides e aumentado a viabilidade dos mesmos.(AU)

Spermatozoa sexing has an important application for livestock production. Results have been published worldwide that demonstrate effectiveness of the flow cytometer sexing process based on sorting sperm with differential DNA content as the X and Y sperm marker. Major improvements in the sexing sorting process as pressure, laser power, extenders had led to the decrease of sperm damage and increase of sperm viability(AU)

Evolução da utilização de espermatozoides sexados / Evolution of sexed spermatozoa utilization

A sexagem de espermatozoides tem relevante aplicação na produção animal. Várias publicações demonstram a eficiência do processo de sexagem por citometria de fluxo que separa os espermatozoides X e Y em função da mensuração do conteúdo de DNA. Importantes modificações no processo de sexagem por citometria de fluxo, tais como pressão, intensidade do laser, diluidores tem levado à diminuição dos danos nos espermatozoides e aumentado a viabilidade dos mesmos.

Spermatozoa sexing has an important application for livestock production. Results have been published worldwide that demonstrate effectiveness of the flow cytometer sexing process based on sorting sperm with differential DNA content as the X and Y sperm marker. Major improvements in the sexing sorting process as pressure, laser power, extenders had led to the decrease of sperm damage and increase of sperm viability

Estudio sobre las diferentes técnicas de sexaje de espermatozoides / Study of different spermatozoa sexing techniques / Estudo sobre as diferentes técnicas de sexagem de espermatozoides

El interés en la selección de individuos de un determinado sexo data de la antigua Grecia; sin embargo, estudios sobre métodos eficaces para la selección espermática son relativamente nuevos. Saber cuál será el sexo de la futura progenie trae al mercado un gran potencial para el mejoramiento genético. Esta selección del sexo es realizada con diversos enfoques; en bovinos para la optimización de animales de sexo deseado, en humanos evitando enfermedades ligadas al sexo y en animales salvajes en cautiverio en la selección del sexo. Con el adviento de las tecnologías como la inseminación artificial y la transferencia de embriones, el sexaje comenzó a tener relevancia en el escenario actual y el costo beneficio pasó a ser significativo. Varias técnicas han sido desarrolladas con el objetivo de mejorarla la pre-selección del sexo, la inmunoseparación, gradientes de densidad y citometria de flujo son algunas de ellas, sin embargo, en la actualidad, la citometria de flujo es la técnica con mejores resultados y la única que permite comercialización de semen sexado. No obstante, el proceso de sexaje aun demanda muchos estudios, debido a los daños ocasionados a los espermatozoides durante el proceso de separación, comprometiendo las tasas de la fertilidad. Esta revisión de literatura viene como una herramienta para el entendimiento del proceso de sexaje y las técnicas utilizadas.(AU)

The interest in sex selection of a particular individuals comes from the ancient greeks, but the study on effective methods of this selection is relatively new. Know future progeny sex brings to market great potential for genetic improvement. Sex selection contributes to cattle for animal optimization of desired sex, to human, preventing diseases sex-linked, or even sex selection of wild animals in captivity. With the biotechnologies advent such as artificial insemination and embryo transfer, sexing begins to have relevance in current scenario and benefit costs will be significant. Several techniques have been developed aiming the best pre-selection of sex, such as immuno separation, density gradient, and flow cytometry that in current world scenario is the technique with best results and only one that allows comercialization of sexed semen. Sexing processes still requiremany studies due to damage to sperm cells during the separation process, compromising fertility results. This literature review comes as a tool for understanding the sexing process and techniques used.(AU)

O interesse na seleção de indivíduos de determinado sexo vem dos antigos gregos, mas o estudo sobre métodos eficazes desta seleção é relativamente novo. Saber qual será o sexo da futura progenie traz ao mercado um grande potencial para o melhoramento genético, A seleção do sexo contribui na bovinocultura para a otimização de animais do sexo desejado, em humanos evitando doenças ligadas ao sexo, ou mesmo na seleção de sexo em animais selvagens de cativeiro. Com o advento das biotecnologias, como a inseminação artificial e transferência de embriões, a sexagem começa a ter relevância no cenário atual e o custo benefício passa a ser significativo. Várias técnicas foram desenvolvidas visando a melhor pré-seleção de sexo, tal como a imuno separação, gradiente de densidade, e a citometria de fluxo que, no atual cenário mundial, é a técnica com os melhores resultados e a única que permite a comercialização do sêmen sexado. Os processos de sexagem ainda demandam muitos estudos, devido aos danos causados aos espermatozoides durante o processo de separação, comprometendo os resultados de fertilidade. Essa revisão de literatura vem como uma ferramenta para o entendimento do processo de sexagem e as técnicas utilizadas.(AU)

Estudio sobre las diferentes técnicas de sexaje de espermatozoides / Study of different spermatozoa sexing techniques / Estudo sobre as diferentes técnicas de sexagem de espermatozoides

El interés en la selección de individuos de un determinado sexo data de la antigua Grecia; sin embargo, estudios sobre métodos eficaces para la selección espermática son relativamente nuevos. Saber cuál será el sexo de la futura progenie trae al mercado un gran potencial para el mejoramiento genético. Esta selección del sexo es realizada con diversos enfoques; en bovinos para la optimización de animales de sexo deseado, en humanos evitando enfermedades ligadas al sexo y en animales salvajes en cautiverio en la selección del sexo. Con el adviento de las tecnologías como la inseminación artificial y la transferencia de embriones, el sexaje comenzó a tener relevancia en el escenario actual y el costo beneficio pasó a ser significativo. Varias técnicas han sido desarrolladas con el objetivo de mejorarla la pre-selección del sexo, la inmunoseparación, gradientes de densidad y citometria de flujo son algunas de ellas, sin embargo, en la actualidad, la citometria de flujo es la técnica con mejores resultados y la única que permite comercialización de semen sexado. No obstante, el proceso de sexaje aun demanda muchos estudios, debido a los daños ocasionados a los espermatozoides durante el proceso de separación, comprometiendo las tasas de la fertilidad. Esta revisión de literatura viene como una herramienta para el entendimiento del proceso de sexaje y las técnicas utilizadas.

The interest in sex selection of a particular individuals comes from the ancient greeks, but the study on effective methods of this selection is relatively new. Know future progeny sex brings to market great potential for genetic improvement. Sex selection contributes to cattle for animal optimization of desired sex, to human, preventing diseases sex-linked, or even sex selection of wild animals in captivity. With the biotechnologies advent such as artificial insemination and embryo transfer, sexing begins to have relevance in current scenario and benefit costs will be significant. Several techniques have been developed aiming the best pre-selection of sex, such as immuno separation, density gradient, and flow cytometry that in current world scenario is the technique with best results and only one that allows comercialization of sexed semen. Sexing processes still requiremany studies due to damage to sperm cells during the separation process, compromising fertility results. This literature review comes as a tool for understanding the sexing process and techniques used.

O interesse na seleção de indivíduos de determinado sexo vem dos antigos gregos, mas o estudo sobre métodos eficazes desta seleção é relativamente novo. Saber qual será o sexo da futura progenie traz ao mercado um grande potencial para o melhoramento genético, A seleção do sexo contribui na bovinocultura para a otimização de animais do sexo desejado, em humanos evitando doenças ligadas ao sexo, ou mesmo na seleção de sexo em animais selvagens de cativeiro. Com o advento das biotecnologias, como a inseminação artificial e transferência de embriões, a sexagem começa a ter relevância no cenário atual e o custo benefício passa a ser significativo. Várias técnicas foram desenvolvidas visando a melhor pré-seleção de sexo, tal como a imuno separação, gradiente de densidade, e a citometria de fluxo que, no atual cenário mundial, é a técnica com os melhores resultados e a única que permite a comercialização do sêmen sexado. Os processos de sexagem ainda demandam muitos estudos, devido aos danos causados aos espermatozoides durante o processo de separação, comprometendo os resultados de fertilidade. Essa revisão de literatura vem como uma ferramenta para o entendimento do processo de sexagem e as técnicas utilizadas.

Fertilization rate and embryo production of superovulated dairy cows after insemination with non-sorted and sex-sorted semen

The aim of this study was to evaluate the fertilization rate of cows that were superovulated and artificially inseminated with sex-sorted semen. Cows were treated with an intravaginal progesterone device plus estradiol benzoate (day 0). Superstimulation treatments began four days after with eight applications of FSH at 12 h intervals. D-Cloprostenol was administered on day 6. Progesterone device was removed on day 7, and LH was administered on day 8. The treatments were divided as follows: NonSx, two AI with non-sorted semen were conducted 12 and 24 h after LH; Sx12&24, two AI with sex-sorted semen were conducted 12 and 24 h after LH; and Sx24&36, two AI with sex-sorted semen were conducted 24 and 36 h after LH. Embryos were recovered on day 16 and were evaluated and classified. Percentage of fertilized embryos tended to be greater for the non-sorted semen than the sex-sorted semen. The number of unfertilized oocytes was smaller when the non-sorted semen was used relative to the sex-sorted semen. There was no difference between the treatments that used sexed semen. In conclusion, the use of sex-sorted semen in superovulated dairy cows results in greater numbers of unfertilized oocytes than non-sorted. However, when only sorted semen is used AI should be performed 24 and 36 h after LH.

Fertilization rate and embryo production of superovulated dairy cows after insemination with non-sorted and sex-sorted semen

The aim of this study was to evaluate the fertilization rate of cows that were superovulated and artificially inseminated with sex-sorted semen. Cows were treated with an intravaginal progesterone device plus estradiol benzoate (day 0). Superstimulation treatments began four days after with eight applications of FSH at 12 h intervals. D-Cloprostenol was administered on day 6. Progesterone device was removed on day 7, and LH was administered on day 8. The treatments were divided as follows: NonSx, two AI with non-sorted semen were conducted 12 and 24 h after LH; Sx12&24, two AI with sex-sorted semen were conducted 12 and 24 h after LH; and Sx24&36, two AI with sex-sorted semen were conducted 24 and 36 h after LH. Embryos were recovered on day 16 and were evaluated and classified. Percentage of fertilized embryos tended to be greater for the non-sorted semen than the sex-sorted semen. The number of unfertilized oocytes was smaller when the non-sorted semen was used relative to the sex-sorted semen. There was no difference between the treatments that used sexed semen. In conclusion, the use of sex-sorted semen in superovulated dairy cows results in greater numbers of unfertilized oocytes than non-sorted. However, when only sorted semen is used AI should be performed 24 and 36 h after LH.(AU)

SEXAGEM E CRIOPRESERVAÇÃO ESPERMÁTICA DE PEQUENOS RUMINANTES UTILIZANDO ANTICORPO MONOCLONAL SEXO-ESPECÍFICO ASSOCIADO AO SISTEMA COMPLEMENTO E DILUENTE DE ORIGEM VEGETAL

A busca pelo melhoramento genético na ovinocaprinocultura através da utilização de biotecnologias reprodutivas como a inseminação artificial associada ao uso de sêmen sexado são de grande valia, uma vez que permite um melhor direcionamento de acordo com os sistemas de produção. Desta forma, o trabalho teve como objetivo avaliar a técnica de imunossexagem por meio do anticorpo monoclonal contra proteína sexo-específica associada à via clássica do complemento e a conservação em diluente de origem vegetal a base de água de coco em pó (ACP101/102c) na criopreservação do sêmen ovino e caprino. O experimento foi realizado no Laboratório de Tecnologia do Sêmen Caprino e Ovino em parceria com as propriedades: Chácara Vetcampo, Complexo HCG e fazenda Guaiúba. O experimento foi realizado em três etapas: (1) Avaliação da influência do ACP101/102c na manutenção da atividade mitocondrial de espermatozoides ovinos criopreservado; (2) Criopreservação de sêmen caprino utilizando ACP101/102c associado ao óleo de coco extra virgem; (3) Criopreservação de espermatozoides ovinos e caprinos imunossexados e diluídos em meio ACP101/102c. As amostras espermáticas foram avaliadas quanto à cinética espermática e quanto as integridades de membranas, morfologia e atividade mitocondrial. Na etapa 1, o meio de conservação ACP- 102c se mostrou eficiente na manutenção da qualidade dos espermatozoiodes ovinos criopreservado. Na etapa 2, em relação à avaliação da motilidade, das integridades de membranas (IMP e IA), da funcionalidade de membrana (HOST) e da atividade mitocondrial (AM) avaliadas após a descongelação não foram observadas diferenças estatísticas significativas entre o controle e as concentrações de óleo de coco 2,5% e 5,0% (P > 0.05). Na etapa 3, na avaliação do sêmen ovino sexado quanto a motilidade espermática e a IMP não foram observadas diferenças estatísticas significativas entre os tratamentos após a sexagem na avaliação dos dados absolutos/brutos (P > 0.05), sendo observada a diferença apenas entre o não sexado (T1) e os sexados (T2 e T3) (P < 0.05). Em relação ao percentual relativo e a morfologia espermática, não foram observadas diferenças estatísticas significativas (P > 0.05). Quanto a avaliação das amostras de sêmen caprino sexado, os parâmetros de motilidade apresentaram-se condizentes com a técnica submetida, entretanto, os dados de IMP não se apresentaram conforme esperado, sendo necessário outras avaliações para melhor avaliação da técnica para esta espécie. Concluindo-se que a utilização do diluente a base de água de coco em pó (ACP101/102c) é eficaz na manutenção da qualidade espermática de ovinos e caprinos criopreservado, bem como a adição do óleo de coco como crioprotetor na espécie caprina, apresentando-se como um diluente alternativo para programas internacionais de inseminação artificial e transferências de embriões. Concluiu-se também que o processo de imunossexagem por meio do anticorpo monoclonal contra proteína sexo-específica (HY) associada à via clássica do sistema complemento e ao uso do diluente à base de água de coco em pó (ACP10/102c) na criopreservação do sêmen mostrou-se eficiente na avaliação in vitro para a espécie ovina.

The search for genetic improvement in ram and goats through the use of reproductive biotechnologies such as artificial insemination associated with the use of sexed semen is of great value, as it allows for better direction according to production systems. Thus, the objective of this work was to evaluate the immunosexing technique using monoclonal antibody against sex-specific protein associated with the classical pathway of complement and preservation in a diluent of vegetable origin based on powdered coconut water (ACP101/102c) in the cryopreservation of the ram and goat semen. The experiment was conducted in Goat and Ram Semen Technology Laboratory in partnership with the properties: Vetcampo Farmhouse, HCG Complex and farm Guaiúba. The experiment was conducted in three stages: (1) Evaluation of the influence of ACP101/102c in the maintenance of the mitochondrial activity of cryopreserved sheep sperm; (2) Cryopreservation of goat semen using ACP101/102c associated with extra virgin coconut oil; (3) Cryopreservation of immunosexed ovine and caprine sperm and diluted in ACP101/102c extender. Sperm samples were evaluated for sperm kinetics and for membrane integrity, morphology and mitochondrial activity. In step 1, the ACP-102c conservation medium proved to be efficient in maintaining the quality of cryopreserved ram sperm. In step 2, regarding the evaluation of the motility of membrane integrity (IMP and IA), the membrane functionality (HOST) and the mitochondrial activity (AM) evaluated after thawing was not observed statistically significant differences between the control and concentrations of coconut oil 2.5% and 5.0% (P> 0.05). In step 3, In the evaluation of sexed ram semen regarding sperm motility and IMP, no statistically significant differences were observed between treatments after sexing in the evaluation of absolute data (P > 0.05), with the difference being observed only between the non-sex (T1) and the sexed (T2 and T3) (P <0.05). Regarding the relative percentage and sperm morphology, no statistically significant differences were observed (P > 0.05). As for the evaluation of sexed goat semen samples, the motility parameters were consistent with the technique submitted, however, the IMP data were not as expected, requiring further evaluations for a better evaluation of the technique for this species. In conclusion, the use of powdered coconut water diluent (ACP101/102c) is effective in maintaining the sperm quality of cryopreserved ram and goats, as well as the addition of coconut oil as a cryoprotectant in goats, as an alternative diluent for international programs of artificial insemination and embryo transfers. It was also concluded that the immunosexing process through monoclonal antibody against sex-specific protein (HY) associated with the classic pathway of the complement system and the use of powdered coconut water diluent (ACP101/102c) in the cryopreservation of semen proved to be efficient in the in vitro evaluation for the ovine species.

Genomic selection of in vitro produced and somatic cell nuclear transfer embryos for rapid genetic improvement in cattle production

This paper provides basic concepts of genomic selection (GS) methods in beef and dairy cattle production in combination with assisted reproductive technologies (ART) such as ovum-pick up and in vitroproduction (OPU-IVP). We first introduce genomic tools and discuss main methods of GS as practiced to-date. The general benefit from GS is that it enables selecting animals accurately early in life using genomic predictions particularly those phenotypes that are very difficult or expensive to measure. While it is known that GS increases genetic gain and profit in conventional cattle breeding, GS is much more desirable when combined with OPU-IVP in cattle production. The expected benefits of GS-OPU-IVP far exceed the benefits achieved by either GS or OPU-IVP alone mainly due to tremendous reduction in generation interval. The genetic improvement will increase even further, if genetic merit of donor cows and bulls used in OPU-IVP for key economic traits are maximal. The paper also highlights some challenges particularly with regard to embryo biopsies and quantity and quality of embryo DNA for whole genome genotyping and ways to overcome difficulties. We briefly discuss the somatic cell nuclear transfer (SCNT) technique in the context of applying GS on fibroblast cell lines from fetuses obtained from OPU-IVP techniques and provide our perspectives on how it might pave way for even more rapid cattle improvement. Main conclusion is that employing genomic selection in ARTs such as OPU-IVP of embryos coupled with embryo sexing and SCNT will lead to rapid dissemination of high genetic merit animals on a scale never been seen before. Finally, the paper outlines current research activities on combined genomic selection and advanced reproductive technologies in the GIFT project consortium (www.gift.ku.dk). (AU)

Genomic selection of in vitro produced and somatic cell nuclear transfer embryos for rapid genetic improvement in cattle production

This paper provides basic concepts of genomic selection (GS) methods in beef and dairy cattle production in combination with assisted reproductive technologies (ART) such as ovum-pick up and in vitroproduction (OPU-IVP). We first introduce genomic tools and discuss main methods of GS as practiced to-date. The general benefit from GS is that it enables selecting animals accurately early in life using genomic predictions particularly those phenotypes that are very difficult or expensive to measure. While it is known that GS increases genetic gain and profit in conventional cattle breeding, GS is much more desirable when combined with OPU-IVP in cattle production. The expected benefits of GS-OPU-IVP far exceed the benefits achieved by either GS or OPU-IVP alone mainly due to tremendous reduction in generation interval. The genetic improvement will increase even further, if genetic merit of donor cows and bulls used in OPU-IVP for key economic traits are maximal. The paper also highlights some challenges particularly with regard to embryo biopsies and quantity and quality of embryo DNA for whole genome genotyping and ways to overcome difficulties. We briefly discuss the somatic cell nuclear transfer (SCNT) technique in the context of applying GS on fibroblast cell lines from fetuses obtained from OPU-IVP techniques and provide our perspectives on how it might pave way for even more rapid cattle improvement. Main conclusion is that employing genomic selection in ARTs such as OPU-IVP of embryos coupled with embryo sexing and SCNT will lead to rapid dissemination of high genetic merit animals on a scale never been seen before. Finally, the paper outlines current research activities on combined genomic selection and advanced reproductive technologies in the GIFT project consortium (www.gift.ku.dk).

Proporção sexual de embriões bovinos PIV 60 D após TETF de acordo com o estágio de desenvolvimento embrionário inovulado / Sex ratio of in vitro produced embryos 60 days after fixed-time embryo transfer according to the developmental stage of the implanted embryo

Devido à ampla e consolidada comercialização de embriões bovinos e também por possuir o maior rebanho comercial do mundo, o Brasil atualmente ocupa posição importante no cenário mundial de produção in vitro (PIV) de embriões. Entretanto, desvios na proporção do sexo têm sido frequentemente relatados para embriões PIV. O presente trabalho teve por objetivo avaliar as taxas de prenhez e sexagem fetal de embriões bovinos PIV aos 60 dias após transferência de embriões em tempo fixo (TETF). Foram utilizadas como doadoras de oócitos fêmeas Bos taurus indicus da raça Nelore (n=2.920) e como receptoras 30.912 vacas mestiças (Bos taurus indicus x Bos taurus taurus). Em 11.341 destas receptoras foi realizada sexagem fetal aos 60 dias de gestação. Não foi observada diferença (P>0,05) para taxa de prenhez, nem para perda embrionária, entre os estágios de desenvolvimento do embrião no momento da TETF (blastocisto inicial, blastocisto e blastocisto expandido). Com relação ao sexo dos produtos obtidos, foi observada maior porcentagem de embriões macho nos estágios de blastocisto e blastocisto expandido, e maior porcentagem de embriões fêmea no estágio de blastocisto inicial. Destaca-se que os resultados obtidos com os embriões nos estágios de desenvolvimento blastocisto e blastocisto expandido são bastante fidedignos, visto que número significativo de embriões nestes estágios foi transferido, e que o resultado obtido com embriões no estágio de desenvolvido blastocisto inicial deve ser visto com ressalva dado o número reduzido de embriões neste estágio...

In view of its wide and consolidated sale of bovine embryos and because it has the world’s largest herd, Brazil currently occupies an important position in the worldwide scenario of in vitro production (IVP) of embryos. However, sex ratio deviations have been frequently reported for IVP embryos. The objective of this study was to evaluate the pregnancy rates and fetal sex of IVP bovine embryos 60 days after fixed-time embryo transfer (FTET). Nellore females (Bos taurus indicus, n=2,920) were used as oocyte donors and 30,912 crossbred cows (Bos taurus indicus x Bos taurus taurus) were used as recipients. Of these recipients, 11,341 were submitted to fetal sexing at 60 days of gestation. No difference (P>0.05) in pregnancy rate or embryo loss was observed between stages of embryo development at the time of FTET (early blastocyst, blastocyst, and expanded blastocyst). Regarding the sex of the products obtained, there was a higher percentage of male embryos in the blastocyst and expanded blastocyst stages and a higher percentage of female embryos in the early blastocyst stage. It should be noted that the results obtained for embryos in the blastocyst and expanded blastocyst stages are highly reliable, considering that a significant number of embryos in these stages were transferred. The results obtained for embryos in the early blastocyst stage should be seen with caution in view of the reduced number of embryos in this stage...

Proporção sexual de embriões bovinos PIV 60 D após TETF de acordo com o estágio de desenvolvimento embrionário inovulado / Sex ratio of in vitro produced embryos 60 days after fixed-time embryo transfer according to the developmental stage of the implanted embryo

Devido à ampla e consolidada comercialização de embriões bovinos e também por possuir o maior rebanho comercial do mundo, o Brasil atualmente ocupa posição importante no cenário mundial de produção in vitro (PIV) de embriões. Entretanto, desvios na proporção do sexo têm sido frequentemente relatados para embriões PIV. O presente trabalho teve por objetivo avaliar as taxas de prenhez e sexagem fetal de embriões bovinos PIV aos 60 dias após transferência de embriões em tempo fixo (TETF). Foram utilizadas como doadoras de oócitos fêmeas Bos taurus indicus da raça Nelore (n=2.920) e como receptoras 30.912 vacas mestiças (Bos taurus indicus x Bos taurus taurus). Em 11.341 destas receptoras foi realizada sexagem fetal aos 60 dias de gestação. Não foi observada diferença (P>0,05) para taxa de prenhez, nem para perda embrionária, entre os estágios de desenvolvimento do embrião no momento da TETF (blastocisto inicial, blastocisto e blastocisto expandido). Com relação ao sexo dos produtos obtidos, foi observada maior porcentagem de embriões macho nos estágios de blastocisto e blastocisto expandido, e maior porcentagem de embriões fêmea no estágio de blastocisto inicial. Destaca-se que os resultados obtidos com os embriões nos estágios de desenvolvimento blastocisto e blastocisto expandido são bastante fidedignos, visto que número significativo de embriões nestes estágios foi transferido, e que o resultado obtido com embriões no estágio de desenvolvido blastocisto inicial deve ser visto com ressalva dado o número reduzido de embriões neste estágio...(AU)

In view of its wide and consolidated sale of bovine embryos and because it has the worlds largest herd, Brazil currently occupies an important position in the worldwide scenario of in vitro production (IVP) of embryos. However, sex ratio deviations have been frequently reported for IVP embryos. The objective of this study was to evaluate the pregnancy rates and fetal sex of IVP bovine embryos 60 days after fixed-time embryo transfer (FTET). Nellore females (Bos taurus indicus, n=2,920) were used as oocyte donors and 30,912 crossbred cows (Bos taurus indicus x Bos taurus taurus) were used as recipients. Of these recipients, 11,341 were submitted to fetal sexing at 60 days of gestation. No difference (P>0.05) in pregnancy rate or embryo loss was observed between stages of embryo development at the time of FTET (early blastocyst, blastocyst, and expanded blastocyst). Regarding the sex of the products obtained, there was a higher percentage of male embryos in the blastocyst and expanded blastocyst stages and a higher percentage of female embryos in the early blastocyst stage. It should be noted that the results obtained for embryos in the blastocyst and expanded blastocyst stages are highly reliable, considering that a significant number of embryos in these stages were transferred. The results obtained for embryos in the early blastocyst stage should be seen with caution in view of the reduced number of embryos in this stage...(AU)

Repeatability of in vitro embryo production in Nellore breed / Repetibilidade na produção in vitro de embriões bovinos da raça Nelore

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.

History and perspectives on bovine embryo transfer

On a worldwide basis, more than 750,000 embryos are produced annually from superovulated donors and more than 450,000 embryos are produced using in vitro techniques. Superovulation and embryo collection are done as frequentlu as every 30 days. Cryopreservation and direct transfer of frozen-thawed embryos results in pregnancy rates near that of fresh embryos. Since the zona pellucida-infact in vivo produced bovine embryo can be made specified pathogen-free by washing procedures, thousands of frozen embryos are marketed internationally on an annual basis. In vitro embryo production is used widely in countries like Brazil and Japan. Polymerase chain reaction (PCR) technology is currentlu being used for sexing embryos and this technology is beginning to be used for "embryo diagnostics" and "embryoi genomics". Sex-sorted bovine semen is also readily available and is being used increasingly, especially for in vitro embryo production.

History and perspectives on bovine embryo transfer

On a worldwide basis, more than 750,000 embryos are produced annually from superovulated donors and more than 450,000 embryos are produced using in vitro techniques. Superovulation and embryo collection are done as frequentlu as every 30 days. Cryopreservation and direct transfer of frozen-thawed embryos results in pregnancy rates near that of fresh embryos. Since the zona pellucida-infact in vivo produced bovine embryo can be made specified pathogen-free by washing procedures, thousands of frozen embryos are marketed internationally on an annual basis. In vitro embryo production is used widely in countries like Brazil and Japan. Polymerase chain reaction (PCR) technology is currentlu being used for sexing embryos and this technology is beginning to be used for "embryo diagnostics" and "embryoi genomics". Sex-sorted bovine semen is also readily available and is being used increasingly, especially for in vitro embryo production.(AU)

Equine assisted reproduction and embryo technologies

Assisted reproductive techniques in the horse have been only recently become available compared to other domestic species, in particular ruminants. The scarce availability of abattoir ovaries and t he lack of interest from horse breeders and breed associations, and the anatomical and physiological differences have been the main reasons for this delay. Progressively though, the technology of oocyte maturation in vitro has been established especially after the application of ICSI to obtain in vitro fertilization. The parallel improvement of oocyte maturation conditions and embryo culture media has increased the rates of embryo development from in vitro matured and in vitro cultured ICSI embryos from 5-10% in the early studies to up to 26% in the latest under experimental conditions with abattoir derived oocytes. In 2003, the birth of the first cloned foal established the technology of somatic cell nuclear transfer. The largest set of data on non-surgical embryo transfer of in vitro produced embryos, from ICSI of in vitro-matured Ovum Pick Up (OPU) oocytes, and from somatic cell nuclear transfer, has been obtained in our laboratory. In the clinical context, where OPU and ICSI are applied for the treatment of female and or male infertility, the yield of embryos has been lower compared to experimental conditions. In conclusion, the basic procedures have been established for the use of assisted reproduction and somatic cell nuclear transfer to a degree suitable for clinical applications and the results have been replicated in several laboratories around the world.

Equine assisted reproduction and embryo technologies

Assisted reproductive techniques in the horse have been only recently become available compared to other domestic species, in particular ruminants. The scarce availability of abattoir ovaries and t he lack of interest from horse breeders and breed associations, and the anatomical and physiological differences have been the main reasons for this delay. Progressively though, the technology of oocyte maturation in vitro has been established especially after the application of ICSI to obtain in vitro fertilization. The parallel improvement of oocyte maturation conditions and embryo culture media has increased the rates of embryo development from in vitro matured and in vitro cultured ICSI embryos from 5-10% in the early studies to up to 26% in the latest under experimental conditions with abattoir derived oocytes. In 2003, the birth of the first cloned foal established the technology of somatic cell nuclear transfer. The largest set of data on non-surgical embryo transfer of in vitro produced embryos, from ICSI of in vitro-matured Ovum Pick Up (OPU) oocytes, and from somatic cell nuclear transfer, has been obtained in our laboratory. In the clinical context, where OPU and ICSI are applied for the treatment of female and or male infertility, the yield of embryos has been lower compared to experimental conditions. In conclusion, the basic procedures have been established for the use of assisted reproduction and somatic cell nuclear transfer to a degree suitable for clinical applications and the results have been replicated in several laboratories around the world.(AU)

Sperm biotechnologies in domestic species: state of the art

Modern livestock breeding is basically dependent on the proper use of semen for artificial insemination of femeles and of other reproductive biotechnologies such as the production of embryos in vitro for embryo transfer (IVP). Both these techniques have made possible not only the wide dissemmation of genetic material onto breeding populations but also anhanced the selection of best sires, owing to the development of better diagnostic techniques for sperm function and of preservation of seminal material over time. Although use ofliquid semen cooled to room temperature, to intermediate temperatures (+16-20ºC) or chilled (+5ºC) dominates in different species cryopreservation is preferred in bovine A1 and it is advancing in other species by the design of new containers, freezing methods and the use of better insemination strategies. Techniques to separate the aliquot of most robust spermatozoa from an ejaculate have shown a renascent particularly for sires with low sperm quality, and technological advances in separating spermatozoa for c hromosomal sex make the technique suitable for commercial use, following applicat ion of novel findings in sperm and seminal plasma (SP) diagnostics and function. Alongside, knowledge of the epigenome and signalling c apabilities of the semen (sperm and SP) call s for further studies regarding transgene production via ICSI for IVP or AI.

Sperm biotechnologies in domestic species: state of the art

Modern livestock breeding is basically dependent on the proper use of semen for artificial insemination (AU) of femeles and of other reproductive biotechnologies such as the production of embryos in vitro for embryo transfer (IVP). Both these techniques have made possible not only the wide dissemmation of genetic material onto breeding populations but also anhanced the selection of best sires, owing to the development of better diagnostic techniques for sperm function and of preservation of seminal material over time. Although use ofliquid semen cooled to room temperature, to intermediate temperatures (+16-20ºC) or chilled (+5ºC) dominates in different species cryopreservation is preferred in bovine A1 and it is advancing in other species by the design of new containers, freezing methods and the use of better insemination strategies. Techniques to separate the aliquot of most robust spermatozoa from an ejaculate have shown a renascent particularly for sires with low sperm quality, and technological advances in separating spermatozoa for c hromosomal sex make the technique suitable for commercial use, following applicat ion of novel findings in sperm and seminal plasma (SP) diagnostics and function. Alongside, knowledge of the epigenome and signalling c apabilities of the semen (sperm and SP) call s for further studies regarding transgene production via ICSI for IVP or AI.(AU)

Produção de embriões bovinos in vitro com sêmen sexado / In vitro production of bovine embryos with sex-sorted semen

Com o advento da sexagem espermática a escolha do sexo da prole se tornou uma grande vantagem, principalmente para os criadores de gado leiteiro, que necessitam de novas matrizes para a manutenção do seu rebanho e para os criadores de gado de corte, que almejam mais machos para a produção de carne. Atualmente, a única técnica conhecida capaz de separar as populações de espermatozoides X e Y de mamíferos, com eficiência e pureza acima de 90%, é a citometria de fluxo. A melhor forma de uso do sêmen sexado é na fertilização in vitro, devido ao reduzido número de espermatozoides disponíveis após a sexagem. Vários fatores podem influenciar os resultados da produção in vitro de embriões na espécie bovina utilizando sêmen sexado; dentre eles destacam-se as baixas taxas de fertilização, de clivagem, de blastocistos, de gestações; capacitação espermática parcial; amostras seminais diluídas e a variação entre touros. Alguns estudos têm sido realizados com o intuito de avaliar as alterações genéticas e epigenéticas decorrentes do uso do sêmen sexado na produção de embriões bovinos in vitro. Portanto, este trabalho tem como objetivo realizar uma revisão de literatura sobre as limitações e as vantagens do uso do sêmen sexado, com ênfase na técnica de citometria de fluxo, durante a produção embrionária in vitro na espécie bovina.

The advent of sex-sorting-sperm sex selection of offspring became a great advantage, especially for dairy farms, which need new heifers for maintenance of his herds, and for beef cattle breeders, who crave more males for meat production. Currently, the only known technique capable of separating the populations of X and Y mammals sperm cells with efficiency and purity above 90% is flow cytometry. The best way of using sexed semen is in vitro fertilization, due to the small number of available sperm after sexing. Several factors can influence the results of in vitro embryos production using bovine sexed semen; among them there are the low rates of fertilization, cleavage, blastocyst, pregnancies, partially sperm capacitation, diluted semen samples and variation among bulls. Several studies have been conducted in order to assess the genetic and epigenetic alterations resulting from the use of sexed semen in the production of bovine embryos in vitro. Therefore, this paper aims to conduct a literature review on the limitations and advantages of using sexed semen, with emphasis on the technique of flow cytometry, during in vitro embryo production in cattle.

Produção de embriões bovinos in vitro com sêmen sexado / In vitro production of bovine embryos with sex-sorted semen

Com o advento da sexagem espermática a escolha do sexo da prole se tornou uma grande vantagem, principalmente para os criadores de gado leiteiro, que necessitam de novas matrizes para a manutenção do seu rebanho e para os criadores de gado de corte, que almejam mais machos para a produção de carne. Atualmente, a única técnica conhecida capaz de separar as populações de espermatozoides X e Y de mamíferos, com eficiência e pureza acima de 90%, é a citometria de fluxo. A melhor forma de uso do sêmen sexado é na fertilização in vitro, devido ao reduzido número de espermatozoides disponíveis após a sexagem. Vários fatores podem influenciar os resultados da produção in vitro de embriões na espécie bovina utilizando sêmen sexado; dentre eles destacam-se as baixas taxas de fertilização, de clivagem, de blastocistos, de gestações; capacitação espermática parcial; amostras seminais diluídas e a variação entre touros. Alguns estudos têm sido realizados com o intuito de avaliar as alterações genéticas e epigenéticas decorrentes do uso do sêmen sexado na produção de embriões bovinos in vitro. Portanto, este trabalho tem como objetivo realizar uma revisão de literatura sobre as limitações e as vantagens do uso do sêmen sexado, com ênfase na técnica de citometria de fluxo, durante a produção embrionária in vitro na espécie bovina.(AU)

The advent of sex-sorting-sperm sex selection of offspring became a great advantage, especially for dairy farms, which need new heifers for maintenance of his herds, and for beef cattle breeders, who crave more males for meat production. Currently, the only known technique capable of separating the populations of X and Y mammals sperm cells with efficiency and purity above 90% is flow cytometry. The best way of using sexed semen is in vitro fertilization, due to the small number of available sperm after sexing. Several factors can influence the results of in vitro embryos production using bovine sexed semen; among them there are the low rates of fertilization, cleavage, blastocyst, pregnancies, partially sperm capacitation, diluted semen samples and variation among bulls. Several studies have been conducted in order to assess the genetic and epigenetic alterations resulting from the use of sexed semen in the production of bovine embryos in vitro. Therefore, this paper aims to conduct a literature review on the limitations and advantages of using sexed semen, with emphasis on the technique of flow cytometry, during in vitro embryo production in cattle.(AU)