Biosensor capability of the endometrium is mediated in part, by altered miRNA cargo from conceptus-derived extracellular vesicles.

We tested the hypothesis that the biosensor capability of the endometrium is mediated in part, by the effect of different cargo contained in the extracellular vesicles secreted by the conceptus during the peri-implantation period of pregnancy. We transferred Bos taurus taurus embryos of different origin, in vivo (high developmental potential (IV)), in vitro (intermediate developmental potential (IVF)), or cloned (low developmental potential (NT)), into Bos taurus indicus recipients. Extracellular vesicles (EVs) recovered from Day 16 conceptus-conditioned medium were characterized and their microRNA (miRNA) cargo sequenced alongside RNA sequencing of their respective endometria. There were substantial differences in the endometrial response to in vivo versus in vitro and in vivo versus cloned conceptuses (1153 and 334DEGs respectively) with limited differences between in vitro Vs cloned conceptuses (36 DEGs). The miRNA cargo contained in conceptus-derived EVs was similar between all three groups (426 miRNA in common). Only 8 miRNAs were different between in vivo and cloned conceptuses, while only 6 miRNAs were different between in vivo and in vitro-derived conceptuses. Treatment of endometrial epithelial cells with mimic or inhibitors for miR-128 and miR-1298 changed the proteomic content of target cells (96 and 85, respectively) of which mRNAs are altered in the endometrium in vivo (PLXDC2, COPG1, HSPA12A, MCM5, TBL1XR1, and TTF). In conclusion, we have determined that the biosensor capability of the endometrium is mediated in part, by its response to different EVs miRNA cargo produced by the conceptus during the peri-implantation period of pregnancy.

Steroidal Regulation of Oviductal microRNAs Is Associated with microRNA-Processing in Beef Cows.

Information on molecular mechanisms through which sex-steroids regulate oviductal function to support early embryo development is lacking. Here, we hypothesized that the periovulatory endocrine milieu affects the miRNA processing machinery and miRNA expression in bovine oviductal tissues. Growth of the preovulatory follicle was controlled to obtain cows that ovulated a small follicle (SF) and subsequently bore a small corpus luteum (CL; SF-SCL) or a large follicle (LF) and large CL (LF-LCL). These groups differed in the periovulatory plasmatic sex-steroid's concentrations. Ampulla and isthmus samples were collected on day four of the estrous cycle. Abundance of DROSHA, DICER1, and AGO4 transcripts was greater in the ampulla than the isthmus. In the ampulla, transcription of these genes was greater for the SF-SCL group, while the opposite was observed in the isthmus. The expression of the 88 most abundant miRNAs and 14 miRNAs in the ampulla and 34 miRNAs in isthmus were differentially expressed between LF-LCL and SF-SCL groups. Integration of transcriptomic and miRNA data and molecular pathways enrichment showed that important pathways were inhibited in the SF-SCL group due to miRNA control. In conclusion, the endocrine milieu affects the miRNA expression in the bovine oviduct in a region-specific manner.

In vitro development and cell allocation after aggregation of syngeneic wild type and fluorescence-expressing bovine cloned embryos / In vitro development and cell allocation after aggregation of syngeneic wild type and fluorescence-expressing bovine cloned embryos

Materials, Methods & Results: In vitro-matured bovine cumulus-oocyte complexes (COC) were manually bisected after cumulus and zona pellucida removal; then, two enucleated hemi-oocytes were paired and fused with either a wild type (WT) or a GFP-expressing (GFP) fetal skin cell at the 11th and 19th passages, respectively. Following chemical activation, reconstructed cloned embryos and zona-free parthenote embryos were in vitro-cultured in microwells, for 7 days, either individually (1 x 100%) or after the aggregation of two structures (2 x 100%) per microwell, as follows: (G1) one WT cloned embryo; (G2) two aggregated WT embryos; (G3) one GFP cloned embryo; (G4) two aggregated GFP embryos; (G5) aggregation of a WT embryo and a GFP embryo; (G6) one parthenote embryo; or (G7) two aggregated parthenote embryos. Fusion (clones), cleavage (Day 2), and blastocyst (Day 7) rates, and embryonic cell allocation were compared by the 2 or Fisher tests. Total cell number (TCN) in blastocysts was analyzed by the Student´s test (P 0.05). Fusion and cleavage rates, and cell allocation were similar between groups. On a per WOW basis, development to the blastocyst stage was similar between groups, except for lower rates of development seen in G3. However, when based on number of embryos per group (one or two), blastocyst development was higher in G1 than all other groups, which were simi

Background: The in vitro production (IVP) of embryos by in vitro fertilization or cloning procedures has been known to cause epigenetic changes in the conceptus that in turn are associated with abnormalities in pre- and postnatal development. Handmade cloning (HMC) procedures and the culture of zona-free embryos in individual microwells provide excellent tools for studies in developmental biology, since embryo development and cell allocation patterns can be evaluated under a wide range of embryo reconstruction arrangements and in in vitro embryo culture conditions. As disturbances in embryonic cell allocation after in vitro embryo manipulations and unusual in vivo conditions during the fi rst third of pregnancy appear to be associated with large offspring, embryo aggregation procedures may allow a compensation for epigenetic defects between aggregated embryos or even may infl uence more favorable cell allocation in embryonic lineages, favoring subsequent development. Thus, the aim of this study was to evaluate in vitro embryo developmental potential and the pattern of cell allocation in blastocysts developed after the aggregation of handmade cloned embryos produced using syngeneic wild type and/or transgenic somatic cells.Materials, Methods & Results: In vitro-matured bovine cumulus-oocyte complexes (COC) were manually bisected after cumulus and zona pellucida removal; then

Morfologia e funcionalidade do pneumócito tipo II e sua relação e variação com a idade gestacional em bovinos

The aim of this study was to characterize the presence of type II pneumocyte and the beginning of the surfactant protein production in bovines to the correlated gestational age. Lungs of fetuses at 4 months gestational age were found to be in the canalicular development phase, without presence of type II pneumocytes or appearance of electrophoretic bands compatible with the presence of the surfactant protein. In fetuses at 5 months of gestational age, the lungs were at the terminal sac phase, with the presence of primitive alveolus, formed by cubical epithelium and areas formed by pneumocytes I and pneumocytes II. No electrophoretic bands compatible to the surfactants proteins were identified. In contrast, lungs of fetuses at 6 months of gestational age showed, development of terminal sac phase, with the presence of type I and type II pneumocytes. In this phase, proteic determination for the SDS - PAGE analysis showed the presence of bands between 26 to 36kDa, demonstrating SP - A production, which is the surfactant protein found in highest amount. From the 7th gestational month on, the phase of terminal sac is more evident and complex, with intense sprouting vascularization. The type I pneumocyte had a more squamous aspect and type II pneumocyte were more globular. In the SDS - PAGE analysis of the bronchial - alveolar wash, surfactant protein bands had been observed with a profile similar to that of the newborn animals. In newborn animals, lungs in alveolar phase showed the development of type I and II pneumocytes. The profile of the bronchial - alveolar wash from the newborn was similar to an adult animal, presenting the same bands in triplets. These results suggest that early born fetuses, from 7 months of pregnancy could have a guarantee of surviving due to possible functionality of respiratory system because the lung presented all necessary features for surphactant protein syntheses. However, new clinical studies related to respiratory system functionality open new venues for newborn bovine's experiments respiratory physiology.

Este estudo objetivou caracterizar a presença de pneumócitos tipo II e o início da produção de lipoproteína surfactante em bovinos, correlacionando a idade gestacional com a síntese de surfactante durante o desenvolvimento fetal. Pulmões de fetos com quatro meses de idade gestacional estavam na fase canalicular de desenvolvimento, sem a presença de pneumócitos tipo II ou bandas eletroforéticas compatíveis com a presença de proteínas surfactante. No 5° mês gestacional, os pulmões dos fetos encontravam-se em fase de saculação terminal, com a presença de alvéolos por epitélio cúbico, com áreas formadas por pneumócitos I e II. Nesse período ainda não foi possível identificar proteína surfactante nos pulmões. Esses órgãos em fetos com seis meses de idade gestacional estavam em fase de saco terminal, com presença de pneumócitos tipo I e II. Nessa fase a análise para determinação protéica do surfactante de feto bovino (SDS - PAGE) demonstrou presença de bandas entre 26 e 36kDa, confirmando produção de SP - A, proteína surfactante encontrada em maior quantidade. A partir do 7° mês gestacional, a fase de saco terminal é mais evidente e complexa, com desenvolvimento de intensa vascularização. O pneumócito tipo I apresentava aspecto mais pavimentoso, e o tipo II apresentava aspecto mais globoso. Na análise SDS - PAGE do lavado bronco - alveolar, bandas de proteína surfactante com aspecto similar ao de animais recém-nascidos foram encontradas. Em recém-nascidos, pulmões na fase alveolar foram observados com pneumócitos tipo I e II característicos. O perfil das bandas do lavado bronco-alveolar dos recém-nascidos foi igual ao de animais adultos. Esses achados sugerem que um animal nascido precocemente, a partir dos sete meses de gestação, teria sua sobrevivência garantida devido a uma possível funcionalidade do sistema respiratório do feto, pois o pulmão possuiria as características necessárias para a síntese de proteínas surfactantes. Entretanto, mais estudos clínicos sobre a funcionalidade do sistema respiratório abrem novas fronteiras de experimentos sobre fisiologia respiratória em recém-nascidos bovinos.

Morfologia e funcionalidade do pneumócito tipo II e sua relação e variação com a idade gestacional em bovinos

The aim of this study was to characterize the presence of type II pneumocyte and the beginning of the surfactant protein production in bovines to the correlated gestational age. Lungs of fetuses at 4 months gestational age were found to be in the canalicular development phase, without presence of type II pneumocytes or appearance of electrophoretic bands compatible with the presence of the surfactant protein. In fetuses at 5 months of gestational age, the lungs were at the terminal sac phase, with the presence of primitive alveolus, formed by cubical epithelium and areas formed by pneumocytes I and pneumocytes II. No electrophoretic bands compatible to the surfactants proteins were identified. In contrast, lungs of fetuses at 6 months of gestational age showed, development of terminal sac phase, with the presence of type I and type II pneumocytes. In this phase, proteic determination for the SDS - PAGE analysis showed the presence of bands between 26 to 36kDa, demonstrating SP - A production, which is the surfactant protein found in highest amount. From the 7th gestational month on, the phase of terminal sac is more evident and complex, with intense sprouting vascularization. The type I pneumocyte had a more squamous aspect and type II pneumocyte were more globular. In the SDS - PAGE analysis of the bronchial - alveolar wash, surfactant protein bands had been observed with a profile similar to that of the newborn animals. In newborn animals, lungs in alveolar phase showed the development of type I and II pneumocytes. The profile of the bronchial - alveolar wash from the newborn was similar to an adult animal, presenting the same bands in triplets. These results suggest that early born fetuses, from 7 months of pregnancy could have a guarantee of surviving due to possible functionality of respiratory system because the lung presented all necessary features for surphactant protein syntheses. However, new clinical studies related to respiratory system functionality open new venues for newborn bovine's experiments respiratory physiology.

Este estudo objetivou caracterizar a presença de pneumócitos tipo II e o início da produção de lipoproteína surfactante em bovinos, correlacionando a idade gestacional com a síntese de surfactante durante o desenvolvimento fetal. Pulmões de fetos com quatro meses de idade gestacional estavam na fase canalicular de desenvolvimento, sem a presença de pneumócitos tipo II ou bandas eletroforéticas compatíveis com a presença de proteínas surfactante. No 5° mês gestacional, os pulmões dos fetos encontravam-se em fase de saculação terminal, com a presença de alvéolos por epitélio cúbico, com áreas formadas por pneumócitos I e II. Nesse período ainda não foi possível identificar proteína surfactante nos pulmões. Esses órgãos em fetos com seis meses de idade gestacional estavam em fase de saco terminal, com presença de pneumócitos tipo I e II. Nessa fase a análise para determinação protéica do surfactante de feto bovino (SDS - PAGE) demonstrou presença de bandas entre 26 e 36kDa, confirmando produção de SP - A, proteína surfactante encontrada em maior quantidade. A partir do 7° mês gestacional, a fase de saco terminal é mais evidente e complexa, com desenvolvimento de intensa vascularização. O pneumócito tipo I apresentava aspecto mais pavimentoso, e o tipo II apresentava aspecto mais globoso. Na análise SDS - PAGE do lavado bronco - alveolar, bandas de proteína surfactante com aspecto similar ao de animais recém-nascidos foram encontradas. Em recém-nascidos, pulmões na fase alveolar foram observados com pneumócitos tipo I e II característicos. O perfil das bandas do lavado bronco-alveolar dos recém-nascidos foi igual ao de animais adultos. Esses achados sugerem que um animal nascido precocemente, a partir dos sete meses de gestação, teria sua sobrevivência garantida devido a uma possível funcionalidade do sistema respiratório do feto, pois o pulmão possuiria as características necessárias para a síntese de proteínas surfactantes. Entretanto, mais estudos clínicos sobre a funcionalidade do sistema respiratório abrem novas fronteiras de experimentos sobre fisiologia respiratória em recém-nascidos bovinos.

Mitochondrial characterization of caracu bulls using molecular marker / Caracterização mitocondrial de bovinos caracu utilizando marcador molecular

This experiment was conducted in order to characterize Caracu bulls from different herds finished in the feedlot regimen, via DNA analyses and the performance and carcass charateristics. Eighty-two male, uncastrated bulls animals of the Caracu breed were evaluated, with a mean initial weight of 381.2 kg and a mean age of 24 months. The animals remained under confinement for 92 days and received a feed consisting of 25% roughage and 75% concentrate. All Caracu bulls analyzed showed B.taurus mtDNA. The stDNA analysis showed two distinct patterns (AB and B): 71 animals had the B allele and 9 had the AB allele. The AB and B animals didn´t showed significant difference for any of the traits evaluated (P>0.05).KEY WORDS: Beef cattle, cytoplasmic characterization, DNA.

Este experimento foi realizado com o objetivo de caracterizar, através de análises de DNA, bovinos Caracu provenientes de propriedades distintas terminados em confinamento e avaliar o crescimento e as características de carcaça. Avaliaram-se 82 animais bovinos machos, inteiros da raça Caracu, com peso médio inicial de 381,2 kg e idade média de 24 meses. Os animais permaneceram 92 dias em confinamento recebendo alimentação composta de 25% de volumoso e 75% de concentrado. Todos os bovinos Caracu analisados apresentaram mtDNA de B.taurus. A análise do stDNA apresentou dois padrões distintos (AB e B), sendo que 71 animais apresentaram o alelo B e 9 animais, o alelo AB. Para todas as características avaliadas, não houve diferença significativa (P>0,05) de crescimento e de carcaça, entre os animais de alelo AB e B. PALAVRAS-CHAVE: Bovino de corte, caracterização citoplasmática, DNA

Characterization of a Dairy Gyr herd with respect to its mitochondrial DNA (mt DNA) origin / Caracterização de um rebanho Gir Leiteiro quanto à origem do DNA mitocondrial (mtDNA)

The Zebu breeds were introduced in Brazil mainly in the last century by imports from the Indian subcontinent. When the Zebu cattle arrived, the national herd suffered a significative change by backcrossing the national cows of taurine origin with Zebu sires. These processes created a polymorphism in the mitochondrial DNA (mtDNA) in the Zebu animals with are in a major part derived from backcrossing and sharing mtDNA of taurine origin. To verify the maternal origin of cows belonging to the Dairy Gyr herd of APTA, Mococa 60 females were analyzed and 33 presented mtDNA from Bos taurus origin and 27 presented mtDNA from Bos indicus origin. None of these animals presented patterns of both mtDNA origins, indicating absence of heteroplasmy for these mitochondrial genotypes.

As raças zebuínas entraram no Brasil principalmente no século passado através de importações do continente indiano. Com a chegada do gado zebu, o rebanho nacional sofreu profunda modificação pelo processo de cruzamento absorvente e foi sendo gradativamente azebuado com a utilização de touros das raças zebuínas. Com isso, criou-se um polimorfismo quanto à origem do DNA mitocondrial (mtDNA) nos animais zebuínos visto grande parte serem oriundos de cruzamentos absorventes a partir de vacas taurinas. Para verificação da origem materna das matrizes do rebanho Gir Leiteiro da APTA, Mococa foram analisadas 60 fêmeas sendo que, 33 apresentaram mtDNA de origem Bos taurus enquanto 27 apresentaram mtDNA de origem Bos indicus . Nenhum animal apresentou os dois genótipos, comprovando assim a ausência de heteroplasmia para este marcador.

MITOCHONDRIAL CHARACTERIZATION OF CARACU BULLS USING MOLECULAR MARKER / CARACTERIZAÇÃO MITOCONDRIAL DE BOVINOS CARACU UTILIZANDO MARCADOR MOLECULAR

This experiment was conducted in order to characterize Caracu bulls from different herds finished in the feedlot regimen, via DNA analyses and the performance and carcass charateristics. Eighty-two male, uncastrated bulls animals of the Caracu breed were evaluated, with a mean initial weight of 381.2 kg and a mean age of 24 months. The animals remained under confinement for 92 days and received a feed consisting of 25% roughage and 75% concentrate. All Caracu bulls analyzed showed B.taurus mtDNA. The stDNA analysis showed two distinct patterns (AB and B): 71 animals had the B allele and 9 had the AB allele. The AB and B animals didn´t showed significant difference for any of the traits evaluated (P>0.05).KEY WORDS: Beef cattle, cytoplasmic characterization, DNA.

Este experimento foi realizado com o objetivo de caracterizar, através de análises de DNA, bovinos Caracu provenientes de propriedades distintas terminados em confinamento e avaliar o crescimento e as características de carcaça. Avaliaram-se 82 animais bovinos machos, inteiros da raça Caracu, com peso médio inicial de 381,2 kg e idade média de 24 meses. Os animais permaneceram 92 dias em confinamento recebendo alimentação composta de 25% de volumoso e 75% de concentrado. Todos os bovinos Caracu analisados apresentaram mtDNA de B.taurus. A análise do stDNA apresentou dois padrões distintos (AB e B), sendo que 71 animais apresentaram o alelo B e 9 animais, o alelo AB. Para todas as características avaliadas, não houve diferença significativa (P>0,05) de crescimento e de carcaça, entre os animais de alelo AB e B. PALAVRAS-CHAVE: Bovino de corte, caracterização citoplasmática, DNA

In vitro development and cell allocation after aggregation of syngeneic wild type and fluorescence-expressing bovine cloned embryos / In vitro development and cell allocation after aggregation of syngeneic wild type and fluorescence-expressing bovine cloned embryos

Materials, Methods & Results: In vitro-matured bovine cumulus-oocyte complexes (COC) were manually bisected after cumulus and zona pellucida removal; then, two enucleated hemi-oocytes were paired and fused with either a wild type (WT) or a GFP-expressing (GFP) fetal skin cell at the 11th and 19th passages, respectively. Following chemical activation, reconstructed cloned embryos and zona-free parthenote embryos were in vitro-cultured in microwells, for 7 days, either individually (1 x 100%) or after the aggregation of two structures (2 x 100%) per microwell, as follows: (G1) one WT cloned embryo; (G2) two aggregated WT embryos; (G3) one GFP cloned embryo; (G4) two aggregated GFP embryos; (G5) aggregation of a WT embryo and a GFP embryo; (G6) one parthenote embryo; or (G7) two aggregated parthenote embryos. Fusion (clones), cleavage (Day 2), and blastocyst (Day 7) rates, and embryonic cell allocation were compared by the 2 or Fisher tests. Total cell number (TCN) in blastocysts was analyzed by the Student´s test (P 0.05). Fusion and cleavage rates, and cell allocation were similar between groups. On a per WOW basis, development to the blastocyst stage was similar between groups, except for lower rates of development seen in G3. However, when based on number of embryos per group (one or two), blastocyst development was higher in G1 than all other groups, which were simi

Background: The in vitro production (IVP) of embryos by in vitro fertilization or cloning procedures has been known to cause epigenetic changes in the conceptus that in turn are associated with abnormalities in pre- and postnatal development. Handmade cloning (HMC) procedures and the culture of zona-free embryos in individual microwells provide excellent tools for studies in developmental biology, since embryo development and cell allocation patterns can be evaluated under a wide range of embryo reconstruction arrangements and in in vitro embryo culture conditions. As disturbances in embryonic cell allocation after in vitro embryo manipulations and unusual in vivo conditions during the fi rst third of pregnancy appear to be associated with large offspring, embryo aggregation procedures may allow a compensation for epigenetic defects between aggregated embryos or even may infl uence more favorable cell allocation in embryonic lineages, favoring subsequent development. Thus, the aim of this study was to evaluate in vitro embryo developmental potential and the pattern of cell allocation in blastocysts developed after the aggregation of handmade cloned embryos produced using syngeneic wild type and/or transgenic somatic cells.Materials, Methods & Results: In vitro-matured bovine cumulus-oocyte complexes (COC) were manually bisected after cumulus and zona pellucida removal; then