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.

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.

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)

Quadro citológico vaginal, concentração plasmática de progesterona durante a gestação e medidas fetais em paca (Cuniculus paca Linnaeus, 1766) / Vaginal citology, serum progesterone concentration during pregnancy and fetal measurements in paca (Cuniculus paca, Linnaeus 1766)

Em 27 pacas (Cuniculus paca Linnaeus, 1766) objetivou-se descrever aos 30, 60 e 90 dias (D) de prenhez diagnosticada por ultrassonografia (US), os tipos celulares do epitélio vaginal em esfregaços vaginais, relatar as condições de abertura da vulva e as características do muco vaginal, determinar a concentração plasmática de progesterona (P4) por radioimunoensaio, e ainda, mensurar por ultrassonografia (US) o diâmetro biparietal (DBP) fetal aos 60 e 90 dias de prenhez. No D30, 40% das amostras exibiram células (com características estrogênicas) superficiais e presença de núcleos nus. Nos D60 e D90, células parabasais, intermediárias, superficiais e naviculares estavam presentes nas mesmas proporções, mas células endocervicais foram descritas em apenas 73,9% e 69% das amostras daqueles dias, respectivamente. No D30 a maior proporção de células naviculares e superficiais diferiu (p < 0,05) em relação aos outros tipos celulares presentes. O muco vaginal apresentou-se cristalino e fluido em 100% e em 70% das fêmeas nos D30 e D60, respectivamente. Observou-se o vestíbulo vaginal aberto em torno de 50% das fêmeas em todos os dias de exames. Valores mínimos detectáveis de P4 foram obtidos em 72% e em 83% das fêmeas, enquanto que as médias das medidas dos DBP foram 1,25 cm (± 0,16) e 2,34 cm (± 0,25) nos D60 e D90, respectivamente. O quadro citológico vaginal nos D30, D60 e D90 e o DBP fornecem elementos que contribuem para diagnóstico de gestação em pacas. A concentração de P4 demonstra a necessidade de maiores estudos da endocrinologia da gestação em pacas.

The objective of this work was to describe in 27 pacas (Cuniculus paca Linnaeus, 1766) at 30, 60 and 90 days (D) of pregnancy diagnosed by ultrasonography (U.S.) the cell types of the vaginal epithelium by vaginal smears, to report the vulva opening condition and the characteristics of vaginal mucus, to determine the progesterone (P4) serum concentration by radioimmunoassay, and also measure by ultrasound (U.S.) the fetuses biparietal diameter (BPD) at 60 and 90 days of pregnancy. At D30, 40% of the smears exhibited surface cells (with estrogenic characteristics) and of naked nuclei. At D60 and D90, parabasal, intermediate, superficial, and navicular cells were present in the same proportions, but endocervical cells were described in only 73.9% and 69% of those day's smears, respectively. At D30 the highest proportion of navicular and surface cells differed (p < 0.05) compared with other cell types. The vaginal mucus was crystalline and fluid in 100% and 70% of females at D30 and D60, respectively. It was observed the vaginal vestibule open in around 50% of females in every day of exams. Minimum detectable P4 serum concentration was obtained on 72% and 83% of females, while the averages of the BPD were 1.25 cm (± 0.16) and 2.34 cm (± 0.25) on D60 and D90, respectively. Vaginal cytology aspects at D30, D60 and D90 and BPD provide elements to contribute to pregnancy diagnosis in paca. P4 concentration shows the need of further endocrinology of pregnancy studies of paca.

Quadro citológico vaginal, concentração plasmática de progesterona durante a gestação e medidas fetais em paca (Cuniculus paca Linnaeus, 1766)

The objective of this work was to describe in 27 pacas (Cuniculus paca Linnaeus, 1766) at 30, 60 and 90 days (D) of pregnancy diagnosed by ultrasonography (U.S.) the cell types of the vaginal epithelium by vaginal smears, to report the vulva opening condition and the characteristics of vaginal mucus, to determine the progesterone (P4) serum concentration by radioimmunoassay, and also measure by ultrasound (U.S.) the fetuses biparietal diameter (BPD) at 60 and 90 days of pregnancy. At D30, 40% of the smears exhibited surface cells (with estrogenic characteristics) and of naked nuclei. At D60 and D90, parabasal, intermediate, superficial, and navicular cells were present in the same proportions, but endocervical cells were described in only 73.9% and 69% of those day's smears, respectively. At D30 the highest proportion of navicular and surface cells differed (p 0.05) compared with other cell types. The vaginal mucus was crystalline and fluid in 100% and 70% of females at D30 and D60, respectively. It was observed the vaginal vestibule open in around 50% of females in every day of exams. Minimum detectable P4 serum concentration was obtained on 72% and 83% of females, while the averages of the BPD were 1.25 cm (± 0.16) and 2.34 cm (± 0.25) on D60 and D90, respectively. Vaginal cytology aspects at D30, D60 and D90 and BPD provide elements to contribute to pregnancy diagnosis in paca. P4 concentration shows the need of further endocrinology of pregnancy studies of paca.

Em 27 pacas (Cuniculus paca Linnaeus, 1766) objetivou-se descrever aos 30, 60 e 90 dias (D) de prenhez diagnosticada por ultrassonografia (US), os tipos celulares do epitélio vaginal em esfregaços vaginais, relatar as condições de abertura da vulva e as características do muco vaginal, determinar a concentração plasmática de progesterona (P4) por radioimunoensaio, e ainda, mensurar por ultrassonografia (US) o diâmetro biparietal (DBP) fetal aos 60 e 90 dias de prenhez. No D30, 40% das amostras exibiram células (com características estrogênicas) superficiais e presença de núcleos nus. Nos D60 e D90, células parabasais, intermediárias, superficiais e naviculares estavam presentes nas mesmas proporções, mas células endocervicais foram descritas em apenas 73,9% e 69% das amostras daqueles dias, respectivamente. No D30 a maior proporção de células naviculares e superficiais diferiu (p 0,05) em relação aos outros tipos celulares presentes. O muco vaginal apresentou-se cristalino e fluido em 100% e em 70% das fêmeas nos D30 e D60, respectivamente. Observou-se o vestíbulo vaginal aberto em torno de 50% das fêmeas em todos os dias de exames. Valores mínimos detectáveis de P4 foram obtidos em 72% e em 83% das fêmeas, enquanto que as médias das medidas dos DBP foram 1,25 cm (± 0,16) e 2,34 cm (± 0,25) nos D60 e D90, respectivamente. O quadro citológico vaginal nos D30, D60 e D90 e o DBP fornecem elementos que contribuem para diagnóstico de gestação em pacas. A concentração de P4 demonstra a necessidade de maiores estudos da endocrinologia da gestação em pacas.

Influência da raça do touro (Bos indicus x Bos taurus) na tolerância ao estresse térmico calórico de embriões bovinos produzidos in vitro / Influence of sire breed (Bos indicus vs Bos taurus) on heat stress tolerance of in vitro-produced bovine embryos

Para melhor compreender as diferenças entre zebuínos e taurinos em relação à resistência ao ETC, objetivou-se verificar se a resistência ao ETC é resultado da contribuição genética do oócito, do espermatozoide ou de ambos. Oócitos de vacas das raças Nelore e mestiças com fenótipo predominante da raça Holandesa preto e branco (mHPB) foram coletados, maturados e fertilizados com espermatozoide de touros das raças Nelore (N), Angus (An), Brahman (Bra) e Gir (Gir). Noventa e seis horas pós-inseminação (hpi), embriões > 16 células foram separados ao acaso em dois grupos: controle e ETC. Embriões do grupo controle foram cultivados a 39 ºC continuamente e do grupo ETC expostos a 41 ºC por 12 horas, retornando a seguir para 39 ºC. Não foi observado efeito do ETC nas raças estudadas, sem redução nas taxas de blastocisto e blastocisto eclodido. As taxas de clivagem e mórula dos embriões mHPB x Gir foram inferiores (p < 0,05) às das demais raças. As raças mHPB x N apresentaram taxas de blastocisto superiores as raças mHPB x An e mHPB x Gir (p < 0,05). Concluiu-se que a contribuição genética do oócito é mais importante do que a do espermatozoide, uma vez que a raça do touro não influenciou a resistência embrionária ao ETC.(AU)

To better understand the differences related to HS resistance between Bos indicus and Bos taurus, we aim to verify if the HS tolerance is due mostly to the genetic contribution from the oocyte, spermatozoa or both. Oocytes from Nelore and crossbreed Holstein cows (cHST) were collected, matured and fertilized with semen from Nelore (N), Angus (An), Brahman (Bra) and Gir (Gir) bulls. Nine six hours post insemination (hpi), > 16 cells embryos were separated in two groups: control and HS. In control group, embryos were cultured at 39 ºC, whereas in the HS group, embryos were subjected to 41 ºC for 12 h, and then returned to 39 ºC. There was no effect of HS on blastocyst and hatched blastocyst rates in all breeds analyzed. The percentage of oocytes that cleaved and reached morula stage was significantly lower (p < 0.05) in cHST x Gir as compared to the other breeds. Additionally, blastocyst rates was higher in cHST x N than in cHST x An and cHST x Gir (p < 0.05). It was concluded that the oocyte is more important than the spermatozoa for the development of thermotolerance, since the breed of the bull did not influence embryo development after HS.(AU)

Influência da raça do touro (Bos indicus x Bos taurus) na tolerância ao estresse térmico calórico de embriões bovinos produzidos in vitro / Influence of sire breed (Bos indicus vs Bos taurus) on heat stress tolerance of in vitro-produced bovine embryos

Para melhor compreender as diferenças entre zebuínos e taurinos em relação à resistência ao ETC, objetivou-se verificar se a resistência ao ETC é resultado da contribuição genética do oócito, do espermatozoide ou de ambos. Oócitos de vacas das raças Nelore e mestiças com fenótipo predominante da raça Holandesa preto e branco (mHPB) foram coletados, maturados e fertilizados com espermatozoide de touros das raças Nelore (N), Angus (An), Brahman (Bra) e Gir (Gir). Noventa e seis horas pós-inseminação (hpi), embriões > 16 células foram separados ao acaso em dois grupos: controle e ETC. Embriões do grupo controle foram cultivados a 39 ºC continuamente e do grupo ETC expostos a 41 ºC por 12 horas, retornando a seguir para 39 ºC. Não foi observado efeito do ETC nas raças estudadas, sem redução nas taxas de blastocisto e blastocisto eclodido. As taxas de clivagem e mórula dos embriões mHPB x Gir foram inferiores (p < 0,05) às das demais raças. As raças mHPB x N apresentaram taxas de blastocisto superiores as raças mHPB x An e mHPB x Gir (p < 0,05). Concluiu-se que a contribuição genética do oócito é mais importante do que a do espermatozoide, uma vez que a raça do touro não influenciou a resistência embrionária ao ETC.

To better understand the differences related to HS resistance between Bos indicus and Bos taurus, we aim to verify if the HS tolerance is due mostly to the genetic contribution from the oocyte, spermatozoa or both. Oocytes from Nelore and crossbreed Holstein cows (cHST) were collected, matured and fertilized with semen from Nelore (N), Angus (An), Brahman (Bra) and Gir (Gir) bulls. Nine six hours post insemination (hpi), > 16 cells embryos were separated in two groups: control and HS. In control group, embryos were cultured at 39 ºC, whereas in the HS group, embryos were subjected to 41 ºC for 12 h, and then returned to 39 ºC. There was no effect of HS on blastocyst and hatched blastocyst rates in all breeds analyzed. The percentage of oocytes that cleaved and reached morula stage was significantly lower (p < 0.05) in cHST x Gir as compared to the other breeds. Additionally, blastocyst rates was higher in cHST x N than in cHST x An and cHST x Gir (p < 0.05). It was concluded that the oocyte is more important than the spermatozoa for the development of thermotolerance, since the breed of the bull did not influence embryo development after HS.

Produção de quimeras embrionárias bovinas mediante a agregação de embriões diploides (Bos indicus) e tetraploides (Bos indicus)

No quimerismo por agregação, células de embriões oriundos de fertilizações distintas se distribuem, aleatoriamente, para formar um único indivíduo. Entretanto, a formação de embriões tetraploides (4n), por eletrofusão, e o posterior quimerismo com um embrião diploide (2n) deve resultar em um embrião quimérico, porém com massa celular interna (MCI) não aleatória, isto é, exclusivamente 2n. Desta forma, a agregação de um embrião zebuíno (4n, genótipo termorresistente) com um taurino (2n, genótipo termossensível) resultaria em uma MCI exclusivamente ?taurina?, porém com o trofectoderma (futuro elemento extraembrionário, isto é, o córion) de maior composição zebuína, podendo constituir um modelo para estudos de adaptação - durante a gestação - deste embrião/feto em clima tropical. O objetivo do presente trabalho foi padronizar a produção de embriões 4n da raça Nelore (Bos indicus) bem como o quimerismo por agregação com embriões mestiço Bos taurus (2n) com Bos indicus (4n). Oócitos de vacas Nelore provenientes de abatedouro foram maturados, fertilizados com sêmen de touros Nelore ou Holandês Preto e Branco e cultivados em meio SOF. Embriões da raça Nelore, em estágio de 2 células (30 h pós-inseminação, hpi) e com o eixo interblastômeros bem definido, foram selecionados para o procedimento de eletrofusão (equipamento ECM 830-BTX, Harvard Apparatus) com o intuito de produzir embriões tetraploides. Para este procedimento, foram testados alguns parâmetros, de acordo com o número de pulsos (1 ou 2), voltagem (0,40; 0,50; 0,75; 1,0; 1,4; 5,0 kV/cm) e duração do eletrochoque (20; 25; 50; 60 ?s). Embriões zebuínos 4n, formados após a eletrofusão, e embriões mestiços taurinos 2n, ambos em estágio de 8 a 16 células (72 hpi), foram submetidos a tratamentos com protease...

The embryonic chimeras production by aggregation allows embryonic cells from two distinct fertilizations to be randomly distributed to compose a single individual. However, the formation of tetraploid embryo (4n) by electrofusion and its subsequent chimerism with a diploid embryo (2n) should result in a chimeric conceptus, whose inner cell mass (ICM) would be entirely 2n. Hence, the aggregation of a zebu embryo (4n, thermotolerant) with a European breed embryo (2n, thermosensitive or ?taurine?) would result in an exclusively taurine ICM, but the trophectoderm (future extraembryonic components, i.e. chorion) would be mostly from zebu embryo, which could support taurine embryo/fetus in a different way - during pregnancy - under tropical environment. The aim of this study was to standardize the production of 4n Nelore embryos (Bos indicus) and the production of embryonic chimeras by aggregation of crossbred Bos taurus (2n) with Bos indicus (4n) embryos. Nelore cows ovaries were collected from abattoir and oocytes were obtained, matured, fertilized with semen from Nelore or Holstein bulls, and cultured in SOF (synthetic oviduct fluid). Two-cell stage Nelore embryos (30 hours post insemination or hpi), with a clearly visible interblastomeric axis, were selected for electrofusion procedure (ECM 830-BTX, Harvard Apparatus) to produce tetraploid embryos. For this purpose, some parameters were tested according to the number of pulses (1 or 2), voltage (40, 50, 75, 100, 140, 500 V) and time length of electroshock (20, 25, 50, 60 ?s). Nelore tetraploid embryos produced after electrofusion and diploid crossbred taurine embryos, both on 8 to 16-cells stage (72 hpi) were subjected to protease treatment to remove the zona pellucida, and subsequently treated with the agglutinant agent...