Estacionalidade e a produção de oócitos e embriões in vitro em vacas Gir leiteiro na região sudeste do Brasil / Seasonality and the production of oocytes and in vitro embryos in Gir dairy cows in the southeast region of Brazil

A eficiência reprodutiva de doadoras de embriões Bos taurus é afetada pelas estações devido ao estresse calórico em países de clima tropical e temperado. O objetivo do estudo foi avaliar o efeito das condições climáticas de verão e inverno na produção de oócitos e embriões in vitro na raça Gir Leiteiro (Bos indicus) em fazendas da região Sudeste do Brasil. Os parâmetros avaliados foram: total de oócitos recuperados (OT), oócitos viáveis (OV) e percentual de oócitos viáveis sobre o total (POV); embriões produzidos (EP) e percentual de embriões produzidos (PEP) sobre o total de estruturas recuperadas pela aspiração folicular guiada por ultrassonografia (OPU). Os oócitos foram obtidos de 153 doadoras pluríparas de diferentes idades e ordens de parição de 10 rebanhos. As médias obtidas em 380 aspirações foliculares (OPU) nos meses de verão foram: OT=14,8; OV=12,6; EP= 3,4; PEP=22,3%, e no inverno de OT=13,4; OV=10,8; EP=3,5; PEP=26,1%, apresentando coeficiente de variação de até 84%, porém sem diferença estatísticas (p<0,05) entre estes períodos, utilizando-se o teste de Kruscal Wallis s (p<0,05). Apenas a percentagem de oócitos viáveis foi maior (p<0,05) no período de verão (POV=84,4 vs. 80,0%), contudo, o total de embriões PIVE foi semelhante nos dois períodos. Conclui-se que a produção de embriões da raça Gir Leiteiro, criadas na região Sudeste do Brasil, não foi afetada pela sazonalidade climática.

The reproductive performance of B.taurus embryo donors is affected in many tropical and temperate countries. The objective of this study was to evaluate the effects of the climatic conditions of summer and winter climatic conditions on the production of oocytes and in vitro embryos in dairy cows of the breed Gir Leiteiro (Bos indicus) at farms in Southeast of the Brazil. The variable answers evaluated were the total oocytes recovered (TO) and percentage of viable oocytes over the total (PVO); embryo production (EP) and percentage of embryos in viable oocytes (PE) over the total of structures recovered by follicular aspiration guided by ultrasonography (OPU). The oocytes analyzed were obtained from 10 herds and 153 pubertal donors of different ages and orders of parturition. The average from 380 ovum pick up (OPU) during summer were: TO=14.8; PVO=12.6; EP=3.4; PE=22.3%, and TO=13.4; PVO=10.8; EP=3.5; PE=26.1%, with variation coefficient of up to 84%, and no differences evaluated through the Kruscal Wallis (p<0.05). Only the percentage of viable oocytes was higher (p<0.05) in the summer period (PVO=84.4 vs. 80.0%). However, the embryo production (IVEP) was similar in both seasons (summer and winter). Therefore, we could conclude that the embryo production of Dairy Gir cows in Southeast of the Brazil was not affected by weather seasonality.

Use of melatonin in the in vitro production of bovine embryos / Uso da melatonina na produção in vitro de embriões bovinos

We aimed to assess the effects of melatonin in the in vitro production of bovine embryos. Our experiment was conducted at the Laboratório de Reprodução Animal of the Universidade Estadual do Maranhão. The cumulus-oocyte complexes (COCs) were distributed among treatments at concentrations of 0, 10-1, 10-3 and 10-5 µMol/L melatonin. Our experiment was further divided into two: the first was to assess the effect of different concentrations of melatonin (treatments) on the maturation rate of COCs, and the second was to assess the effects of melatonin treatments on the in vitro production of bovine embryos. The results from the first experiment demonstrated no significant difference between the in vitro maturation rate of the cultivated COCs in treatments with melatonin. In the second experiment, however, melatonin treatments yielded statistically higher cleavage, morula and blastocyst rates in the 10-5 µM group (52.9%, 52.9%, and 35.3%, respectively), and lower rates in the 10-1 µM group (19.5%, 19.5% and 7.8%, respectively), compared to the others. The control group (no melatonin) and the 10-3 µM group showed similar results. We concluded that supplementation of melatonin in the in vitro maturation medium resulted in no improvement in the oocyte maturation rate, but in the in vitro production of embryos at different concentrations, the 10-5 µM group displayed better results, but with no improvement in the variables (P < 0.05).(AU)

Objetivou-se avaliar os efeitos da melatonina na produção in vitro de embriões bovinos. O experimento foi conduzido no Laboratório de Reprodução Animal da Universidade Estadual do Maranhão. Os complexos cumulus-oócitos (CCOs) foram distribuídos entre os tratamentos 0, 10-1, 10-3 e 10-5 µmol/L de melatonina. A avaliação foi dividida em dois experimentos, onde o primeiro avaliou o efeito dessas diferentes concentrações de melatonina (tratamentos) sobre a taxa de maturação dos CCOs e o segundo, o efeito desses tratamentos com melatonina sobre a produção in vitro de embriões bovinos. Os resultados no primeiro experimento demonstraram não haver diferença significativa na taxa de maturação in vitro dos CCOs cultivados no tratamento com melatonina. No entanto, o tratamento com melatonina no segundo experimento, as taxas de clivagens, mórulas e blastocistos, o grupo 10-5 µM foi estatisticamente superior (52,9%, 52,9% e 35,3%, respectivamente) e o grupo 10-1 µM inferior (19,5%, 19,5% e 7,8%, respectivamente) aos outros grupos. O grupo controle (sem melatonina) e o grupo 10-3 µM obtiveram resultados semelhantes. Concluiu-se que a suplementação da melatonina no meio de maturação in vitro não evidenciou melhoras na taxa de maturação dos oócitos, porém na produção in vitro de embriões em diferentes concentrações, o grupo 10-5 µM apresentou melhores resultados mesmo não havendo melhorias nas variáveis (P<0,05).(AU)

Estacionalidade e a produção de oócitos e embriões in vitro em vacas Gir leiteiro na região sudeste do Brasil / Seasonality and the production of oocytes and in vitro embryos in Gir dairy cows in the southeast region of Brazil

A eficiência reprodutiva de doadoras de embriões Bos taurus é afetada pelas estações devido ao estresse calórico em países de clima tropical e temperado. O objetivo do estudo foi avaliar o efeito das condições climáticas de verão e inverno na produção de oócitos e embriões in vitro na raça Gir Leiteiro (Bos indicus) em fazendas da região Sudeste do Brasil. Os parâmetros avaliados foram: total de oócitos recuperados (OT), oócitos viáveis (OV) e percentual de oócitos viáveis sobre o total (POV); embriões produzidos (EP) e percentual de embriões produzidos (PEP) sobre o total de estruturas recuperadas pela aspiração folicular guiada por ultrassonografia (OPU). Os oócitos foram obtidos de 153 doadoras pluríparas de diferentes idades e ordens de parição de 10 rebanhos. As médias obtidas em 380 aspirações foliculares (OPU) nos meses de verão foram: OT=14,8; OV=12,6; EP= 3,4; PEP=22,3%, e no inverno de OT=13,4; OV=10,8; EP=3,5; PEP=26,1%, apresentando coeficiente de variação de até 84%, porém sem diferença estatísticas (p<0,05) entre estes períodos, utilizando-se o teste de Kruscal Wallis s (p<0,05). Apenas a percentagem de oócitos viáveis foi maior (p<0,05) no período de verão (POV=84,4 vs. 80,0%), contudo, o total de embriões PIVE foi semelhante nos dois períodos. Conclui-se que a produção de embriões da raça Gir Leiteiro, criadas na região Sudeste do Brasil, não foi afetada pela sazonalidade climática.(AU)

The reproductive performance of B.taurus embryo donors is affected in many tropical and temperate countries. The objective of this study was to evaluate the effects of the climatic conditions of summer and winter climatic conditions on the production of oocytes and in vitro embryos in dairy cows of the breed Gir Leiteiro (Bos indicus) at farms in Southeast of the Brazil. The variable answers evaluated were the total oocytes recovered (TO) and percentage of viable oocytes over the total (PVO); embryo production (EP) and percentage of embryos in viable oocytes (PE) over the total of structures recovered by follicular aspiration guided by ultrasonography (OPU). The oocytes analyzed were obtained from 10 herds and 153 pubertal donors of different ages and orders of parturition. The average from 380 ovum pick up (OPU) during summer were: TO=14.8; PVO=12.6; EP=3.4; PE=22.3%, and TO=13.4; PVO=10.8; EP=3.5; PE=26.1%, with variation coefficient of up to 84%, and no differences evaluated through the Kruscal Wallis (p<0.05). Only the percentage of viable oocytes was higher (p<0.05) in the summer period (PVO=84.4 vs. 80.0%). However, the embryo production (IVEP) was similar in both seasons (summer and winter). Therefore, we could conclude that the embryo production of Dairy Gir cows in Southeast of the Brazil was not affected by weather seasonality.(AU)

Use of melatonin in the in vitro production of bovine embryos / Uso da melatonina na produção in vitro de embriões bovinos

We aimed to assess the effects of melatonin in the in vitro production of bovine embryos. Our experiment was conducted at the Laboratório de Reprodução Animal of the Universidade Estadual do Maranhão. The cumulus-oocyte complexes (COCs) were distributed among treatments at concentrations of 0, 10-1, 10-3 and 10-5 µMol/L melatonin. Our experiment was further divided into two: the first was to assess the effect of different concentrations of melatonin (treatments) on the maturation rate of COCs, and the second was to assess the effects of melatonin treatments on the in vitro production of bovine embryos. The results from the first experiment demonstrated no significant difference between the in vitro maturation rate of the cultivated COCs in treatments with melatonin. In the second experiment, however, melatonin treatments yielded statistically higher cleavage, morula and blastocyst rates in the 10-5 µM group (52.9%, 52.9%, and 35.3%, respectively), and lower rates in the 10-1 µM group (19.5%, 19.5% and 7.8%, respectively), compared to the others. The control group (no melatonin) and the 10-3 µM group showed similar results. We concluded that supplementation of melatonin in the in vitro maturation medium resulted in no improvement in the oocyte maturation rate, but in the in vitro production of embryos at different concentrations, the 10-5 µM group displayed better results, but with no improvement in the variables (P < 0.05).

Objetivou-se avaliar os efeitos da melatonina na produção in vitro de embriões bovinos. O experimento foi conduzido no Laboratório de Reprodução Animal da Universidade Estadual do Maranhão. Os complexos cumulus-oócitos (CCOs) foram distribuídos entre os tratamentos 0, 10-1, 10-3 e 10-5 µmol/L de melatonina. A avaliação foi dividida em dois experimentos, onde o primeiro avaliou o efeito dessas diferentes concentrações de melatonina (tratamentos) sobre a taxa de maturação dos CCOs e o segundo, o efeito desses tratamentos com melatonina sobre a produção in vitro de embriões bovinos. Os resultados no primeiro experimento demonstraram não haver diferença significativa na taxa de maturação in vitro dos CCOs cultivados no tratamento com melatonina. No entanto, o tratamento com melatonina no segundo experimento, as taxas de clivagens, mórulas e blastocistos, o grupo 10-5 µM foi estatisticamente superior (52,9%, 52,9% e 35,3%, respectivamente) e o grupo 10-1 µM inferior (19,5%, 19,5% e 7,8%, respectivamente) aos outros grupos. O grupo controle (sem melatonina) e o grupo 10-3 µM obtiveram resultados semelhantes. Concluiu-se que a suplementação da melatonina no meio de maturação in vitro não evidenciou melhoras na taxa de maturação dos oócitos, porém na produção in vitro de embriões em diferentes concentrações, o grupo 10-5 µM apresentou melhores resultados mesmo não havendo melhorias nas variáveis (P<0,05).

Substituição parcial do soro fetal bovino durante cultivo in vitro reduz a concentração de fosfolipídios em embriões bovinos produzidos in vitro / Partial replacement of fetal bovine serum during in vitro culture decreases phospholipid content in in vitro produced bovine embryos

Soro fetal bovino (SFB) e albumina sérica bovina (BSA) são componentes importantes do cultivo in vitro (CIV) de embriões bovinos, porém são frequentemente associados ao acúmulo excessivo de lipídios, podendo prejudicar o desenvolvimento embrionário. Este estudo teve como objetivo substituir parcialmente o SFB por BSA V FAF durante o CIV de embriões bovinos, avaliar a produção embrionária e quantificar os lipídios dos embriões, SFB e dos meios de cultivo. Para isto, os embriões desenvolveram em meios de cultivo suplementados com 10% de SFB (SFB10%) ou 5% de SFB e 0.03g de BSA V FAF (SFB5%/BSA). O conteúdo lipídico foi avaliado por UHPLC-MS/MS. A análise estatística foi feita utilizando teste t e ANOVA. A substituição parcial de SFB por BSA V FAF não alterou a produção embrionária. Nos dois grupos foram identificados 10 fosfolipídios e três deles, DOPC (p=0,037), POPG (p=0,046) e C24: 1-SM (p=0,009), apresentaram menores concentrações no meio SFB5%/BSA. Os fosfolipídios identificados nos embriões coincidem com os encontrados no SFB e meios de cultivo e quatro deles DOPC (p=0,013), DPPC (p=0,004), POPG (p=0,05) e C24:1-SM (p=0,003) diminuíram a concentração com a redução do SFB. A substituição parcial do SFB diminui a concentração de fosfolipídios sem prejudicar a produção embrionária, sugerindo uma melhora nas técnicas relacionadas ao cultivo in vitro.(AU)

Fetal bovine serum (FBS) and bovine serum albumin (BSA) are important components during bovine embryo in vitro culture (IVC), but they are associated with excess of embryonic lipid, which might impair embryo development. This study aimed to partially replace FBS by BSA V FAF during bovine IVC, evaluate embryo production and quantify the phospholipid content in produced embryos, SFB and IVC medium. The embryos were in vitro cultured in medium supplied with 10% of FBS (FBS10%) or with 5% of FBS plus 0.03 g BSA V FAF (FBS5%/BSA). The lipid content was evaluated using UHPLC-MS/MS and statistical analysis was performed using t-test and ANOVA. The partial replacement of FBS by BSA V FAF did not alter embryo production. Ten phospholipids were identified in both groups and three of them, DOPC (p=0.037), POPG (p=0.046) and C24: 1-SM (p=0.009) presented lower concentration in FBS5%/BSA culture medium. The phospholipids identified on embryos matches with those found on SFB and culture medium and four of them DOPC (p=0.013), DPPC (p=0.004), POPG (p=0.05) and C24:1- SM (p=0.003) reduced its concentration when FBS was reduced. Theses founds shown that the FBS partial replacement reduces phospholipids content in embryos but do not decrease embryo production, suggesting a technical improvement.(AU)

Substituição parcial do soro fetal bovino durante cultivo in vitro reduz a concentração de fosfolipídios em embriões bovinos produzidos in vitro / Partial replacement of fetal bovine serum during in vitro culture decreases phospholipid content in in vitro produced bovine embryos

Soro fetal bovino (SFB) e albumina sérica bovina (BSA) são componentes importantes do cultivo in vitro (CIV) de embriões bovinos, porém são frequentemente associados ao acúmulo excessivo de lipídios, podendo prejudicar o desenvolvimento embrionário. Este estudo teve como objetivo substituir parcialmente o SFB por BSA V FAF durante o CIV de embriões bovinos, avaliar a produção embrionária e quantificar os lipídios dos embriões, SFB e dos meios de cultivo. Para isto, os embriões desenvolveram em meios de cultivo suplementados com 10% de SFB (SFB10%) ou 5% de SFB e 0.03g de BSA V FAF (SFB5%/BSA). O conteúdo lipídico foi avaliado por UHPLC-MS/MS. A análise estatística foi feita utilizando teste t e ANOVA. A substituição parcial de SFB por BSA V FAF não alterou a produção embrionária. Nos dois grupos foram identificados 10 fosfolipídios e três deles, DOPC (p=0,037), POPG (p=0,046) e C24: 1-SM (p=0,009), apresentaram menores concentrações no meio SFB5%/BSA. Os fosfolipídios identificados nos embriões coincidem com os encontrados no SFB e meios de cultivo e quatro deles DOPC (p=0,013), DPPC (p=0,004), POPG (p=0,05) e C24:1-SM (p=0,003) diminuíram a concentração com a redução do SFB. A substituição parcial do SFB diminui a concentração de fosfolipídios sem prejudicar a produção embrionária, sugerindo uma melhora nas técnicas relacionadas ao cultivo in vitro.

Fetal bovine serum (FBS) and bovine serum albumin (BSA) are important components during bovine embryo in vitro culture (IVC), but they are associated with excess of embryonic lipid, which might impair embryo development. This study aimed to partially replace FBS by BSA V FAF during bovine IVC, evaluate embryo production and quantify the phospholipid content in produced embryos, SFB and IVC medium. The embryos were in vitro cultured in medium supplied with 10% of FBS (FBS10%) or with 5% of FBS plus 0.03 g BSA V FAF (FBS5%/BSA). The lipid content was evaluated using UHPLC-MS/MS and statistical analysis was performed using t-test and ANOVA. The partial replacement of FBS by BSA V FAF did not alter embryo production. Ten phospholipids were identified in both groups and three of them, DOPC (p=0.037), POPG (p=0.046) and C24: 1-SM (p=0.009) presented lower concentration in FBS5%/BSA culture medium. The phospholipids identified on embryos matches with those found on SFB and culture medium and four of them DOPC (p=0.013), DPPC (p=0.004), POPG (p=0.05) and C24:1- SM (p=0.003) reduced its concentration when FBS was reduced. Theses founds shown that the FBS partial replacement reduces phospholipids content in embryos but do not decrease embryo production, suggesting a technical improvement.

Fatores que influenciam a taxa de concepção e perda gestacional de embriões produzidos in vitro na raça Girolando / Factors influencing the conception rate and pregnancy loss in Girolando embryos produced in vitro

A produção in vitro de embriões é de fundamental importância para a pecuária, entretanto ainda é uma biotecnologia com resultados variados sendo, portanto, necessário estudar formas de melhorar. Objetivou-se com este trabalho avaliar se a categoria das receptoras, estação do ano e estágio de desenvolvimento embrionário afetam as taxas de concepção e as perdas gestacionais em receptoras de embriões da raça Girolando produzidos in vitro e transferidos em tempo fixo (TETF). O experimento foi realizado em uma fazenda leiteira comercial, localizada na região de Uberlândia – MG. Foram avaliadas 1.017 TETF. As receptoras foram divididas em vacas em lactação (n=618), vacas secas (n=44) e novilhas (n=355). As estações do ano foram agrupadas em verão (primavera e verão) e inverno (outono e inverno). Os embriões, todos com qualidade morfológica grau 1, foram agrupados de acordo com seu estágio de desenvolvimento em: Grupo 1 (n=216) - blastocisto inicial (BI) e blastocisto (BL) e Grupo 2 (n=801) - blastocisto expandido (BX), blastocisto em eclosão (BN) e blastocisto eclodido (BE). Foi detectado efeito da categoria da receptora na taxa de concepção (p<0,001) e na perda de gestação (p<0,003). O verão afetou negativamente (p<0,044) a taxa de concepção. O Grupo 2 teve mais sucesso na manutenção da gestação (p<0,025). Conclui-se que os três fatores avaliados interferem nas taxas de concepção e/ou perda gestacional e devem ser considerados nos programas de transferência de embriões da raça Girolando.

The in vitro production of embryos is fundamental importance for livestock, although it is still a biotechnology with varieds results than expected, being therefore necessary study ways to improve its rates. The objective of this study was to evaluate if the recipient category, season and stage of embryo development affect conception rates and gestational losses in recipients of girolando’s embryos produced in vitro and transferred at fixed time (ETFT). The experiment was carried out in a commercial dairy farm, located in the Uberlândia - MG region. A total of 1,017 TETF were evaluated. The recipients were divided into lactating cows (n=618), dry cows (n=44) and heifers (n=355). The seasons were grouped in summer (spring and summer) and winter (autumn and winter). The embryos were grouped in Group 1 (n=216) - initial blastocyst (BI) and blastocyst (BL) and Group 2 (n=801) - expanded blastocyst (BX), hatching blastocyst (BH) and hatched (BE). Receptor category effect was detected at conception rate (p<0.001) and loss of gestation (p<0.003). The summer affected negatively (p<0.044) the conception rate. Group 2 was more successful in maintaining gestation (p<0.025). It is concluded that the three evaluated factors interfere in conception rates and / or gestational loss and should be considered in the genetic improvement programs embryo transfer of the Girolando breed.

Fatores que influenciam a taxa de concepção e perda gestacional de embriões produzidos in vitro na raça Girolando / Factors influencing the conception rate and pregnancy loss in Girolando embryos produced in vitro

A produção in vitro de embriões é de fundamental importância para a pecuária, entretanto ainda é uma biotecnologia com resultados variados sendo, portanto, necessário estudar formas de melhorar. Objetivou-se com este trabalho avaliar se a categoria das receptoras, estação do ano e estágio de desenvolvimento embrionário afetam as taxas de concepção e as perdas gestacionais em receptoras de embriões da raça Girolando produzidos in vitro e transferidos em tempo fixo (TETF). O experimento foi realizado em uma fazenda leiteira comercial, localizada na região de Uberlândia MG. Foram avaliadas 1.017 TETF. As receptoras foram divididas em vacas em lactação (n=618), vacas secas (n=44) e novilhas (n=355). As estações do ano foram agrupadas em verão (primavera e verão) e inverno (outono e inverno). Os embriões, todos com qualidade morfológica grau 1, foram agrupados de acordo com seu estágio de desenvolvimento em: Grupo 1 (n=216) - blastocisto inicial (BI) e blastocisto (BL) e Grupo 2 (n=801) - blastocisto expandido (BX), blastocisto em eclosão (BN) e blastocisto eclodido (BE). Foi detectado efeito da categoria da receptora na taxa de concepção (p<0,001) e na perda de gestação (p<0,003). O verão afetou negativamente (p<0,044) a taxa de concepção. O Grupo 2 teve mais sucesso na manutenção da gestação (p<0,025). Conclui-se que os três fatores avaliados interferem nas taxas de concepção e/ou perda gestacional e devem ser considerados nos programas de transferência de embriões da raça Girolando.(AU)

The in vitro production of embryos is fundamental importance for livestock, although it is still a biotechnology with varieds results than expected, being therefore necessary study ways to improve its rates. The objective of this study was to evaluate if the recipient category, season and stage of embryo development affect conception rates and gestational losses in recipients of girolandos embryos produced in vitro and transferred at fixed time (ETFT). The experiment was carried out in a commercial dairy farm, located in the Uberlândia - MG region. A total of 1,017 TETF were evaluated. The recipients were divided into lactating cows (n=618), dry cows (n=44) and heifers (n=355). The seasons were grouped in summer (spring and summer) and winter (autumn and winter). The embryos were grouped in Group 1 (n=216) - initial blastocyst (BI) and blastocyst (BL) and Group 2 (n=801) - expanded blastocyst (BX), hatching blastocyst (BH) and hatched (BE). Receptor category effect was detected at conception rate (p<0.001) and loss of gestation (p<0.003). The summer affected negatively (p<0.044) the conception rate. Group 2 was more successful in maintaining gestation (p<0.025). It is concluded that the three evaluated factors interfere in conception rates and / or gestational loss and should be considered in the genetic improvement programs embryo transfer of the Girolando breed.(AU)

A importância dos estudos transcriptômicos de gametas e embriões para o avanço e consolidação da Produção In Vitro de Embriões (PIVE) em bubalinos na Amazônia / The importance of the transcriptomic studies of gametes and embryos for the advance and consolidation of In Vitro Production (IVP) in buffaloes in the Amazon

A produção de búfalos está se expandindo na Amazônia, sendo assim o uso de biotecnologias reprodutivas em programas de melhoramento genético tornou-se uma estratégia para melhorar a produtividade dos rebanhos sem a necessidade de aumentar as áreas de pastagens. A Produção In Vitro de Embriões (PIVE) é uma das biotecnologias disponíveis, a qual é contiuamente aperfeiçoada através da pesquisa de novos componentes para os meios de cultivo e da pesquisa de protocolos espécie específicos que resultem em maior número de embriões de boa qualidade. Contudo, a falta de informações sobre os requerimentos metabólicos de gametas e embriões têm dificultado a consolidação de protocolos de PIVE específicos para búfalos. Neste artigo discutimos as abordagens transcriptômicas que estão ajudando a preencher esta falta de informações, as limitações destas abordagens e os tópicos de pesquisa que ainda precisam ser investigados em búfalos.(AU)

Buffalo production is expanding in the Amazon region therefore the use of reproductive biotechnologies in breeding programs became a strategy to improve the herd productivity without a need for increasing pasture areas. In Vitro Production (IVP) is one of the available biotechnologies, which is continuously being improved through the research of novel components of culture media and the research of specie-specific protocols to obtain an increased number of good quality embryos. However, the lack of information about the metabolic requirements of gametes and embryos have delayed the consolidation of buffalos IVP specific protocols. Herein, we discussed the transcriptomic approaches that are helping to fill the lack of information, the limitations of these approaches and the research topics that still needs further investigations in buffaloes.(AU)

A importância dos estudos transcriptômicos de gametas e embriões para o avanço e consolidação da Produção In Vitro de Embriões (PIVE) em bubalinos na Amazônia / The importance of the transcriptomic studies of gametes and embryos for the advance and consolidation of In Vitro Production (IVP) in buffaloes in the Amazon

A produção de búfalos está se expandindo na Amazônia, sendo assim o uso de biotecnologias reprodutivas em programas de melhoramento genético tornou-se uma estratégia para melhorar a produtividade dos rebanhos sem a necessidade de aumentar as áreas de pastagens. A Produção In Vitro de Embriões (PIVE) é uma das biotecnologias disponíveis, a qual é contiuamente aperfeiçoada através da pesquisa de novos componentes para os meios de cultivo e da pesquisa de protocolos espécie específicos que resultem em maior número de embriões de boa qualidade. Contudo, a falta de informações sobre os requerimentos metabólicos de gametas e embriões têm dificultado a consolidação de protocolos de PIVE específicos para búfalos. Neste artigo discutimos as abordagens transcriptômicas que estão ajudando a preencher esta falta de informações, as limitações destas abordagens e os tópicos de pesquisa que ainda precisam ser investigados em búfalos.

Buffalo production is expanding in the Amazon region therefore the use of reproductive biotechnologies in breeding programs became a strategy to improve the herd productivity without a need for increasing pasture areas. In Vitro Production (IVP) is one of the available biotechnologies, which is continuously being improved through the research of novel components of culture media and the research of specie-specific protocols to obtain an increased number of good quality embryos. However, the lack of information about the metabolic requirements of gametes and embryos have delayed the consolidation of buffalo’s IVP specific protocols. Herein, we discussed the transcriptomic approaches that are helping to fill the lack of information, the limitations of these approaches and the research topics that still needs further investigations in buffaloes.

In vitro production of bovine embryos: cumulus/granulosa cell gene expression patterns point to early atresia as beneficial for oocyte competence

In vitro production (IVP) of bovine embryos has become widespread technology implemented in cattle breeding and production. Here, we review novel data on cumulus/granulosa cell gene expression, as determined by RNAseq on cellular material from pooled follicular fluids at the single animal level, and relate these finding to previous data on oocyte developmental competence and ultrastructure. The cumulus/granulosa cell gene expression patterns indicate that early follicular atresia is associated with increased blastocyst yield and this hypothesis is supported by previous data on oocyte competence and ultrastructure.

Application of integrative genomics and systems biology to conventional and in vitro reproductive traits in cattle

Assisted reproductive technologies (ARTs) have a strong impact on breeding especially when coupled with genomic selection (GS). The routine implementation of in vitro production (IVP) and GS of embryos before embryo transfer (ET) in breeding companies is not yet possible. Improvement of oocyte donor and embryo recipient quality is needed to make realistic a commercialization of these procedures in the near future. A better understanding of both biological mechanisms and molecular markers associated to IVP-ET related traits is necessary to improve the prediction of donor and recipient cow quality for IVP procedures. The huge amount of data generated from highthroughputtechnologies has a tremendous impact in the search for biomarkers of complex traits. This paper reviews integrative genomics and systms biology approaches as applied to both Bos indicus and Bos taurus cattle reproduction by both conventional and ARTs such as OPU-IVP. The integration of systems biology information across different biological layers generates a complete view of the different molecular networks that control complex traits and can provide a strong contribution to the understanding traits related to ARTs.

Intracytoplasmic sperm injection after vitrifcation of immature oocytes in follicular fluid increases bovine embryo production

Background: Despite the low efficiency caused by its harmful effects, vitrification is the technique of choice for oocyte cryopeservation, especially at the germinal vesicle (GV) stage. This enables the banking of female gametes without linkage to the male genotype. Follicular fluid (FF), in vivo, is known to provide an adequate environment to the immature oocyte. The intra-cytoplasmic sperm injection (ICSI), by the other hand, can be used to bypass any sperm penetration disorder, including the ones caused by cryopreservation. This study aimed to evaluate oocyte vitrification in FF based solution, and to asses ICSI efficiency in the fertilization of vitrified/warmed bovine GV oocytes. Material, Methods & Results: Follicles of 2-8 mm in diameter were aspirated from bovine ovaries obtained from a slaughterhouse, selected and maintained into FF from aspiration, until their allocation in the experimental groups. The FF used to prepare the vitrification solution was centrifuged, heat inactivated, filtered through a 0.22 mm pore and stored at -20°C. Oocyte vitrification was done into one of these three solutions: The standard solution TCM-Hepes (TH-Vitri) was compared to a totally FF based solution (FF-Vitri), and to a 50:50 (v/v) mix of both solutions (TH:FF-Vitri). Oocytes were submitted to in vitroembryo production in order to assess embryo production efficiency. A second set of experiments using the FF-Vitri solution compared IVF versus ICSI. With basis on cleaved structures, the morula + blastocyst rate obtained in the Fresh Control (43.9%) was similar to FF-Vitri (31.1%). Conversely, the TH-Vitri (15.7%) and the TH:FF-Vitri (20.4%) rates were significantly lower than the Fresh Control. ICSI showed a positive effect in comparison with IVF. The embryo development rate of Vitri-IVF (18.8%) was the lowest, whereas Vitri-ICSI (37.3%) was similar to the Fresh-IVF (43.9%), but lower than the Fresh-ICSI (57.8%).[...]

Intracytoplasmic Sperm Injection after Vitrification of Immature Oocytes in Follicular Fluid Increases Bovine Embryo Production

Background: Despite the low efficiency caused by its harmful effects, vitrification is the technique of choice for oocyte cryopeservation, especially at the germinal vesicle (GV) stage. This enables the banking of female gametes without linkage to the male genotype. Follicular fluid (FF), in vivo, is known to provide an adequate environment to the immature oocyte. The intra-cytoplasmic sperm injection (ICSI), by the other hand, can be used to bypass any sperm penetration disorder, including the ones caused by cryopreservation. This study aimed to evaluate oocyte vitrification in FF based solution, and to asses ICSI efficiency in the fertilization of vitrified/warmed bovine GV oocytes.Material, Methods & Results: Follicles of 2-8 mm in diameter were aspirated from bovine ovaries obtained from a slaughterhouse, selected and maintained into FF from aspiration, until their allocation in the experimental groups. The FF used to prepare the vitrification solution was centrifuged, heat inactivated, filtered through a 0.22 mm pore and stored at -20C. Oocyte vitrification was done into one of these three solutions: The standard solution TCM-Hepes (TH-Vitri) was compared to a totally FF based solution (FF-Vitri), and to a 50:50 (v/v) mix of both solutions (TH:FF-Vitri). Oocytes were submitted to in vitro embryo production in order to assess embryo production efficiency. A second set

Intracytoplasmic sperm injection after vitrifcation of immature oocytes in follicular fluid increases bovine embryo production

Background: Despite the low efficiency caused by its harmful effects, vitrification is the technique of choice for oocyte cryopeservation, especially at the germinal vesicle (GV) stage. This enables the banking of female gametes without linkage to the male genotype. Follicular fluid (FF), in vivo, is known to provide an adequate environment to the immature oocyte. The intra-cytoplasmic sperm injection (ICSI), by the other hand, can be used to bypass any sperm penetration disorder, including the ones caused by cryopreservation. This study aimed to evaluate oocyte vitrification in FF based solution, and to asses ICSI efficiency in the fertilization of vitrified/warmed bovine GV oocytes. Material, Methods & Results: Follicles of 2-8 mm in diameter were aspirated from bovine ovaries obtained from a slaughterhouse, selected and maintained into FF from aspiration, until their allocation in the experimental groups. The FF used to prepare the vitrification solution was centrifuged, heat inactivated, filtered through a 0.22 mm pore and stored at -20°C. Oocyte vitrification was done into one of these three solutions: The standard solution TCM-Hepes (TH-Vitri) was compared to a totally FF based solution (FF-Vitri), and to a 50:50 (v/v) mix of both solutions (TH:FF-Vitri). Oocytes were submitted to in vitroembryo production in order to assess embryo production efficiency. A second set of experiments using the FF-Vitri solution compared IVF versus ICSI. With basis on cleaved structures, the morula + blastocyst rate obtained in the Fresh Control (43.9%) was similar to FF-Vitri (31.1%). Conversely, the TH-Vitri (15.7%) and the TH:FF-Vitri (20.4%) rates were significantly lower than the Fresh Control. ICSI showed a positive effect in comparison with IVF. The embryo development rate of Vitri-IVF (18.8%) was the lowest, whereas Vitri-ICSI (37.3%) was similar to the Fresh-IVF (43.9%), but lower than the Fresh-ICSI (57.8%).[...](AU)

In vitro production of bovine embryos: cumulus/granulosa cell gene expression patterns point to early atresia as beneficial for oocyte competence

In vitro production (IVP) of bovine embryos has become widespread technology implemented in cattle breeding and production. Here, we review novel data on cumulus/granulosa cell gene expression, as determined by RNAseq on cellular material from pooled follicular fluids at the single animal level, and relate these finding to previous data on oocyte developmental competence and ultrastructure. The cumulus/granulosa cell gene expression patterns indicate that early follicular atresia is associated with increased blastocyst yield and this hypothesis is supported by previous data on oocyte competence and ultrastructure.(AU)

Application of integrative genomics and systems biology to conventional and in vitro reproductive traits in cattle

Assisted reproductive technologies (ARTs) have a strong impact on breeding especially when coupled with genomic selection (GS). The routine implementation of in vitro production (IVP) and GS of embryos before embryo transfer (ET) in breeding companies is not yet possible. Improvement of oocyte donor and embryo recipient quality is needed to make realistic a commercialization of these procedures in the near future. A better understanding of both biological mechanisms and molecular markers associated to IVP-ET related traits is necessary to improve the prediction of donor and recipient cow quality for IVP procedures. The huge amount of data generated from highthroughputtechnologies has a tremendous impact in the search for biomarkers of complex traits. This paper reviews integrative genomics and systms biology approaches as applied to both Bos indicus and Bos taurus cattle reproduction by both conventional and ARTs such as OPU-IVP. The integration of systems biology information across different biological layers generates a complete view of the different molecular networks that control complex traits and can provide a strong contribution to the understanding traits related to ARTs.(AU)

A new simple and reliable vitrification device based on Hollow Fiber Vitrification (HFV) method evaluated using IVP bovine embryos

A new vitrification device based on hollow fiber vitrification (HFV) was constructed using a glass capillary, which lead to simplified construction process and increased practicality of the device. The hollow fiber was attached to heat-pulled tip of the glass capillary using forceps. A protective sheath fitted on the capillary provided protection for the cellulose triacetate hollow fiber with loaded embryos and allowed safe storage in liquid nitrogen for long periods of time (2-12 month), transfer between tanks with liquid nitrogen and transportation within these tanks. No embryos were lost in the process. The device was tested using seven-day-old and eight-day-old IVP bovine blastocysts and expanded blastocysts as a model. Obtained survival (90% at 24 h post warming) and hatching rates (62% at 72 h post warming) of day 7 blastocysts and expanded blastocysts were comparable to those gained using various vitrification carriers. Vitrified embryos did not show an increase in the number of cells with damaged membrane or a decrease in total cell number per embryos in comparison to their non-vitrified counterparts. Day 7 and 8 expanded blastocysts did not differ significantly in terms of survival at 24 (97.01 vs. 97.50%) and 48 h post warming (95.52 vs. 95%), but showed significantly higher survival and hatching rates than day 7 and 8 blastocysts. These results indicated that high and repeatable survival rates can be obtained by selection of IVP bovine embryos at the developmental stage of expanded blastocyst for HFV. Further modification of the method may be required to achieve high and stable results with different developmental stages of IVP bovine embryo. The vitrification device presented in the current article may contribute to wider application of HFV method in livestock production.

A new simple and reliable vitrification device based on Hollow Fiber Vitrification (HFV) method evaluated using IVP bovine embryos

A new vitrification device based on hollow fiber vitrification (HFV) was constructed using a glass capillary, which lead to simplified construction process and increased practicality of the device. The hollow fiber was attached to heat-pulled tip of the glass capillary using forceps. A protective sheath fitted on the capillary provided protection for the cellulose triacetate hollow fiber with loaded embryos and allowed safe storage in liquid nitrogen for long periods of time (2-12 month), transfer between tanks with liquid nitrogen and transportation within these tanks. No embryos were lost in the process. The device was tested using seven-day-old and eight-day-old IVP bovine blastocysts and expanded blastocysts as a model. Obtained survival (90% at 24 h post warming) and hatching rates (62% at 72 h post warming) of day 7 blastocysts and expanded blastocysts were comparable to those gained using various vitrification carriers. Vitrified embryos did not show an increase in the number of cells with damaged membrane or a decrease in total cell number per embryos in comparison to their non-vitrified counterparts. Day 7 and 8 expanded blastocysts did not differ significantly in terms of survival at 24 (97.01 vs. 97.50%) and 48 h post warming (95.52 vs. 95%), but showed significantly higher survival and hatching rates than day 7 and 8 blastocysts. These results indicated that high and repeatable survival rates can be obtained by selection of IVP bovine embryos at the developmental stage of expanded blastocyst for HFV. Further modification of the method may be required to achieve high and stable results with different developmental stages of IVP bovine embryo. The vitrification device presented in the current article may contribute to wider application of HFV method in livestock production.(AU)

Efeito da melatonina na maturação in vitro de oócitos caprinos / Effect of melatonin on in vitro maturation of goat oocytes

To evaluate the effect of melatonin on in vitro maturation of goat oocytes, 12 females were used asoocyte donor. They were subjected to ovarian stimulation and ovum pickup by laparotomy. Oocytes wereevaluated and allocated into three groups of in vitro maturation: M0 (oocytes matured in TCM199 andsupplements); M10 and M50 (oocytes matured in M0 medium supplemented with 10 μM and 50 μM melatonin,respectively). All groups were subjected to the cell incubator for 24 hours at 38.5°C with a humidifiedatmosphere containing 5% CO2. For data analysis, Chi-square test was used, with a probability of 5% and acorrelation test. In conclusion, melatonin reduced in vitro maturation rates of goat oocytes.(AU)