Production of a cloned calf from a fetal fibroblast cell line

The present study examined the in vitro and in vivo development of bovine nuclear-transferred embryos. A bovine fetal fibroblast culture was established and used as nucleus donor. Slaughterhouse oocytes were matured in vitro for 18 h before enucleation. Enucleated oocytes were fused with fetal fibroblasts with an electric stimulus and treated with cytochalasin D and cycloheximide for 1 h followed by cycloheximide alone for 4 h. Reconstructed embryos were cultured for 7-9 days and those which developed to blastocysts were transferred to recipient cows. Of 191 enucleated oocytes, 83 (43.5%) were successfully fused and 24 (28.9%) developed to blastocysts. Eighteen freshly cloned blastocysts were transferred to 14 recipients, 5 (27.8%) of which were pregnant on day 35 and 3 (16.7%) on day 90. Of the three cows that reached the third trimester, one recipient died of hydrallantois 2 months before term, one aborted fetus was recovered at 8 months of gestation, and one delivered by cesarian section a healthy cloned calf. Today, the cloned calf is 15 months old and presents normal body development (378 kg) and sexual behavior (libido and semen characteristics).

Alternative parameters for echocardiographic assessment of fetal diastolic function

Alternative methods to assess ventricular diastolic function in the fetus are proposed. Fetal myocardial hypertrophy in maternal diabetes was used as a model of decreased left ventricular compliance (LVC), and fetal respiratory movements as a model of increased LVC. Comparison of three groups of fetuses showed that, in 10 fetuses of diabetic mothers (FDM) with septal hypertrophy (SH), the mean excursion index of the septum primum (EISP) (ratio between the linear excursion of the flap valve and the left atrial diameter) was 0.36 ± 0.09, in 8 FDM without SH it was 0.51 ± 0.09 (P = 0.001), and in the 8 normal control fetuses (NCF) it was 0.49 ± 0.12 (P = 0.003). In another study, 28 fetuses in apnea had a mean EISP of 0.39 ± 0.05 which increased to 0.57 ± 0.07 during respiration (P < 0.001). These two studies showed that the mobility of the septum primum was reduced when LVC was decreased and was increased when LVC was enhanced. Mean pulmonary vein pulsatility was higher in 14 FDM (1.83 ± 1.21) than in 26 NCF (1.02 ± 0.31; P = 0.02). In the same fetuses, mean left atrial shortening was decreased (0.40 ± 0.11) in relation to NCF (0.51 ± 0.09; P = 0.011). These results suggest that FDM may have a higher preload than normal controls, probably as a result of increased myocardial mass and LV hypertrophy. Prenatal assessment of LV diastolic function by fetal echocardiography should include analysis of septum primum mobility, pulmonary vein pulsatility, and left atrial shortening.

Cholesterol induces fetal rat enterocyte death in culture

The effect of cholesterol on fetal rat enterocytes and IEC-6 cells (line originated from normal rat small intestine) was examined. Both cells were cultured in the presence of 20 to 80 µM cholesterol for up to 72 h. Apoptosis was determined by flow cytometric analysis and fluorescence microscopy. The expression of HMG-CoA reductase and peroxisome proliferator-activated receptor gamma (PPARgamma) was measured by RT-PCR. The addition of 20 µM cholesterol reduced enterocyte proliferation as early as 6 h of culture. Reduction of enterocyte proliferation by 28 and 41% was observed after 24 h of culture in the presence and absence of 10% fetal calf serum, respectively, with the effect lasting up to 72 h. Treatment of IEC-6 cells with cholesterol for 24 h raised the proportion of cells with fragmented DNA by 9.7% at 40 µM and by 20.8% at 80 µM. When the culture period was extended to 48 h, the effect of cholesterol was still more pronounced, with the percent of cells with fragmented DNA reaching 53.5% for 40 µM and 84.3% for 80 µM. Chromatin condensation of IEC-6 cells was observed after treatment with cholesterol even at 20 µM. Cholesterol did not affect HMG-CoA reductase expression. A dose-dependent increase in PPARgamma expression in fetal rat enterocytes was observed. The expression of PPAR-gamma was raised by 7- and 40-fold, in the presence and absence of fetal calf serum, respectively, with cholesterol at 80 mM. The apoptotic effect of cholesterol on enterocytes was possibly due to an increase in PPARgamma expression.

Relationships between fetal body weight of Wistar rats at term and the extent of skeletal ossification

We investigated the relationship between fetal body weight at term (pregnancy day 21) and the extent of ossification of sternum, metacarpus, metatarsus, phalanges (proximal, medial and distal) of fore- and hindlimbs and cervical and coccygeal vertebrae in Wistar rats. The relationships between fetal body weight and sex, intrauterine position, uterine horn, horn size, and litter size were determined using historical control data (7594 fetuses; 769 litters) of untreated rats. Relationships between body weight and degree of ossification were examined in a subset of 1484 historical control fetuses (154 litters) which were subsequently cleared and stained with alizarin red S. Fetal weight was independent of horn size, uterine horn side (left or right) or intrauterine position. Males were heavier than females and fetal weight decreased with increasing litter size. Evaluation of the skeleton showed that ossification of sternum, metacarpus and metatarsus was extensively complete and independent of fetal weight on pregnancy day 21. In contrast, the extent of ossification of fore- and hindlimb phalanges and of cervical and sacrococcygeal vertebrae was dependent on fetal body weight. The strongest correlation between body weight and degree of ossification was found for hindlimb, medial and proximal phalanges. Our data therefore suggest that, in full-term rat fetuses (day 21), reduced ossification of sternum, metacarpus and metatarsus results from a localized impairment of bone calcification (i.e., a malformation or variation) rather than from general growth retardation and that ossification of hindlimb (medial and proximal) phalanges is a good indicator of treatment-induced fetal growth retardation.

A connection between extracellular matrix and hormonal signals during the development of the human fetal adrenal gland

The human adrenal cortex, involved in adaptive responses to stress, body homeostasis and secondary sexual characters, emerges from a tightly regulated development of a zone-specific secretion pattern during fetal life. Its development during fetal life is critical for the well being of pregnancy, the initiation of delivery, and even for an adequate adaptation to extra-uterine life. As early as from the sixth week of pregnancy, the fetal adrenal gland is characterized by a highly proliferative zone at the periphery, a concentric migration accompanied by cell differentiation (cortisol secretion) and apoptosis in the central androgen-secreting fetal zone. After birth, a strong reorganization occurs in the adrenal gland so that it better fulfills the newborn's needs, with aldosterone production in the external zona glomerulosa, cortisol secretion in the zona fasciculata and androgens in the central zona reticularis. In addition to the major hormonal stimuli provided by angiotensin II and adrenocorticotropin, we have tested for some years the hypotheses that such plasticity may be under the control of the extracellular matrix. A growing number of data have been harvested during the last years, in particular about extracellular matrix expression and its putative role in the development of the human adrenal cortex. Laminin, collagen and fibronectin have been shown to play important roles not only in the plasticity of the adrenal cortex, but also in cell responsiveness to hormones, thus clarifying some of the unexplained observations that used to feed controversies.

Development of fetal nicotine and muscarinic receptors in utero

The role of acetylcholine in the central and peripheral nervous systems is well established in adults. Cholinergic modulation of vascular functions and body fluid balance has been extensively studied. In the embryo-fetus, cholinergic receptors are widespread in the peripheral and central systems, including smooth muscle and the epithelial lining of the cardiovascular, digestive, and urinary systems, as well as in the brain. Fetal nicotine and muscarinic receptors develop in a pattern (e.g., amount and distribution) related to gestational periods. Cholinergic mechanisms have been found to be relatively intact and functional in the control of vascular homeostasis during fetal life in utero at least during the last third of gestation. This review focuses on the development of fetal nicotine and muscarinic receptors, and provides information indicating that central cholinergic systems are well developed in the control of fetal blood pressure and body fluid balance before birth. Therefore, the development of cholinergic systems in utero plays an important role in fetal vascular regulation, gastrointestinal motility, and urinary control.

Fetal development of regulatory mechanisms for body fluid homeostasis

The balance of body fluids is critical to health and the development of diseases. Although quite a few review papers have shown that several mechanisms, including hormonal and behavioral regulation, play an important role in body fluid homeostasis in adults, there is limited information on the development of regulatory mechanisms for fetal body fluid balance. Hormonal, renal, and behavioral control of body fluids function to some extent in utero. Hormonal mechanisms including the renin-angiotensin system, aldosterone, and vasopressin are involved in modifying fetal renal excretion, reabsorption of sodium and water, and regulation of vascular volume. In utero behavioral changes, such as fetal swallowing, have been suggested to be early functional development in response to dipsogens. Since diseases, such as hypertension, can be traced to fetal origin, it is important to understand the development of fetal regulatory mechanisms for body fluid homeostasis in this early stage of life. This review focuses on fetal hormonal, behavioral, and renal development related to regulation of body fluids in utero.

Maternal undernutrition and the offspring kidney: from fetal to adult life

Maternal dietary protein restriction during pregnancy is associated with low fetal birth weight and leads to renal morphological and physiological changes. Different mechanisms can contribute to this phenotype: exposure to fetal glucocorticoid, alterations in the components of the renin-angiotensin system, apoptosis, and DNA methylation. A low-protein diet during gestation decreases the activity of placental 11ß-hydroxysteroid dehydrogenase, exposing the fetus to glucocorticoids and resetting the hypothalamic-pituitary-adrenal axis in the offspring. The abnormal function/expression of type 1 (AT1R) or type 2 (AT2R) AngII receptors during any period of life may be the consequence or cause of renal adaptation. AT1R is up-regulated, compared with control, on the first day after birth of offspring born to low-protein diet mothers, but this protein appears to be down-regulated by 12 days of age and thereafter. In these offspring, AT2R expression differs from control at 1 day of age, but is also down-regulated thereafter, with low nephron numbers at all ages: from the fetal period, at the end of nephron formation, and during adulthood. However, during adulthood, the glomerular filtration rate is not altered, due to glomerulus and podocyte hypertrophy. Kidney tubule transporters are regulated by physiological mechanisms; Na+/K+-ATPase is inhibited by AngII and, in this model, the down-regulated AngII receptors fail to inhibit Na+/K+-ATPase, leading to increased Na+ reabsorption, contributing to the hypertensive status. We also considered the modulation of pro-apoptotic and anti-apoptotic factors during nephrogenesis, since organogenesis depends upon a tight balance between proliferation, differentiation and cell death.

Plasma concentrations and placental immunostaining of interleukin-10 and tumornecrosis factor-α as predictors of alterations in the embryo-fetal organism and the placental development of diabetic rats

Interleukin-10 (IL-10) appears to be the key cytokine for the maintenance of pregnancy and inhibits the secretion of inflammatory cytokines such as tumor necrosis factor-α (TNF-α). However, there are no studies evaluating the profile of these cytokines in diabetic rat models. Thus, our aim was to analyze IL-10 and TNF-α immunostaining in placental tissue and their respective concentrations in maternal plasma during pregnancy in diabetic rats in order to determine whether these cytokines can be used as predictors of alterations in the embryo-fetal organism and in placental development. These parameters were evaluated in non-diabetic (control; N = 15) and Wistar rats with streptozotocin (STZ)-induced diabetes (N = 15). At term, the dams (100 days of life) were killed under anesthesia and plasma and placental samples were collected for IL-10 and TNF-α determinations by ELISA and immunohistochemistry, respectively. The reproductive performance was analyzed. Plasma IL-10 concentrations were reduced in STZ rats compared to controls (7.6 ± 4.5 vs 20.9 ± 8.1 pg/mL). The placental scores of immunostaining intensity did not differ between groups (P > 0.05). Prevalence analysis showed that the IL-10 expression followed TNF-α expression, showing a balance between them. STZ rats also presented impaired reproductive performance and reduced plasma IL-10 levels related to damage during early embryonic development. However, the increased placental IL-10 as a compensatory mechanism for the deficit of maternal regulation permitted embryo development. Therefore, the data suggest that IL-10 can be used as a predictor of changes in the embryo-fetal organism and in placental development in pregnant diabetic rats.

Modificações dos componentes de parede celular do melão tipo Galia durante a maturação

O presente estudo caracterizou quantitativa e qualitativamente as mudanças nos componentes de parede celular do melão tipo Galia, híbrido Nun 1380, associadas ao processo de maturação. Os frutos foram avaliados em cinco estádios de maturação (I-V). O material de parede celular e suas frações foram determinados por gravimetria. Determinou-se o conteúdo de açúcares neutros na fração hemicelulósica e no resíduo celulósico, o teor de ácidos urônicos e o grau de esterificação na fração de substâncias pécticas, e o teor de cálcio total e ligado. A fração de substâncias pécticas foi submetida à cromatografia de filtração em gel e os açúcares neutros da fração hemicelulósica foram analisados por cromatografia a gás. O conteúdo do material de parede celular (MPC) mostrou pouca variação durante a maturação. O conteúdo de ácidos urônicos da fração péctica e o conteúdo de açúcares neutros na fração hemicelulósica (ramnose, arabinose e glicose) apresentaram tendência de redução com o avanço da maturação do fruto. Praticamente não houve variação para o grau de esterificação (% de esterificação) da fração de substâncias pécticas e para o teor de cálcio ligado. Houve redução no teor de celulose durante a maturação. A cromatografia em gel não revelou tendência de despolimerização das substâncias pécticas.

Ciclo de maturação e produção de etileno de tomates (Lycopersicon esculentum, Mill.) transgênicos

Objetivou-se estudar o comportamento fisiológico de frutos de tomateiro (Lycopersicon esculentum, Mill.), da cv. Kadá, transformados geneticamente, via Agrobacterium tumefaciens, com o clone de DNA pMEL1, em orientação antisenso, e de frutos desta mesma cultivar, não transformados. O estudo fisiológico foi realizado avaliando-se a duração do ciclo de maturação dos frutos, amadurecidos na planta e após a colheita no estádio verde-maduro, e sua produção de etileno. Os frutos transformados amadurecidos na planta tiveram um ciclo total médio de 27 dias, enquanto os amadurecidos após a colheita, tiveram este intervalo prolongado a 50 dias. Ao contrário, os tomates não transformados apresentaram um ciclo de maturação mais acelerado quando colhidos no estádio verde-maduro, em relação aos frutos amadurecidos nas plantas. Os valores foram, em média, de 20 e 30 dias, respectivamente. Estes resultados estão correlacionados com as variações na produção de etileno observada nos dois genótipos. Frutos não transformados produziram, em média, 10,46 nL de etileno.g-1.h-1, enquanto os transgênicos tiveram sua produção de etileno diminuída para 0,13 nL.g-1.h-1. Pode-se concluir, então, que a redução da produção de etileno, verificada nos tomates transformados, é necessária, mas não é suficiente para prolongar o ciclo de maturação e aumentar a durabilidade dos frutos. Para que isto ocorra, é necessário que se proceda à colheita dos tomates no estádio verde-maduro.

Ensaio tecnológico e sensorial de soja [Glycine max (L.) Merrill] enlatada em estádios verdes e no estádio da maturação de colheita

Foi estudada a viabilidade de consumo da cultivar de soja IAC PL-1 enlatada como grão verde e sua melhor época de colheita para enlatamento. Para tanto procederam-se cinco colheitas a partir do 48º dia após a floração (DAF) até a extinção da coloração verde. O processamento iniciou-se pelo branqueamento das vagens, debulha e enlatamento. Aos cinco lotes obtidos adicionou-se um sexto lote para estudar o efeito do armazenamento. Para qualificar os seis lotes de grãos enlatados foram efetuadas medidas de peso, cor, textura e características do líquido de enlatamento. Foram realizados estudos das propriedades sensoriais dos grãos enlatados para dimensionar a aceitação pela degustação e aparência. O enlatamento foi otimizado para 121ºC com tempo de esterilização em torno de 4 minutos. A maturidade fisiológica dos grãos ocorreu entre o 61º ao 64º DAF. O processo térmico conservou a cor verde dos grãos enlatados e não induziu perda expressiva da massa dos grãos; a textura firme dos grãos aumentou com a maturação. Os provadores demonstraram boa aceitação dos produtos e não ocorreu preferência em relação aos estádios de maturação. O trabalho conclui que grãos verdes de soja IAC PL-1 proporcionam enlatados com boas características técnicas e gustativas. Foi observado que grãos colhidos no ponto convencional de maturação, armazenados e enlatados obtiveram boa aceitação gustativa, concluindo-se que a cultivar IAC PL-1 é também adequada para o consumo e enlatada após o armazenamento.

Aspectos químicos e bioquímicos de leguminosas enlatadas em diferentes estádios de maturação

Este trabalho teve como objetivo estudar a composição química e aspectos bioquímicos dos grãos de soja cultivar IAC PL-1 e de guandu cultivar IAC Fava Larga, crus e pós-enlatamento em diferentes estádios de maturação avaliando os efeitos do processamento sobre os grãos dos dois cultivares. A composição química dos grãos crus , principalmente no último estádio verde, e na maturação de colheita foi de modo geral semelhante. O enlatamento conservou 95% e 98% do total das proteínas dos grãos de soja e guandu, respectivamente. Nos enlatados de soja obteve-se a inativação da atividade de lectinas. Os processamentos térmicos utilizados para os enlatamentos, 121ºC por 6 a 7 minutos para a soja e 5 a 6 minutos para o guandu foram suficientes para eliminar 83% da atividade dos inibidores de tripsina da soja e do guandu. A digestibilidade da proteína do guandu enlatado (62%) foi inferior em relação à soja enlatada (78%). Com exceção do ácido glutâmico, prolina, lisina e histidina, os demais aminoácidos do grão de soja enlatado colhido no 64º DAF tiveram seus conteúdos iguais aos enlatados do 85º DAF pós-armazenados e pós-macerados. Os teores de aminoácidos dos grãos de guandu enlatados no 62º DAF, com exceção do ácido glutâmico e fenilalanina, foram iguais àqueles presentes na última colheita (92º DAF). A metionina disponível no grão de soja não se modificou com a evolução da maturação, porém a do guandu se elevou no 92º DAF e o processo de enlatamento reduziu a metionina disponível da soja apenas no 55º e 64º DAF e do guandu no 57º e 92º DAF. Rafinose e estaquiose nos grãos de soja estão mais elevadas no estádio verde, e nos grãos de guandu, apenas a estaquiose está mais elevada no estádio verde. O processo de enlatamento provocou um pequeno decréscimo nestes dois açúcares da soja e guandu nos dois últimos estádios de maturação estudados.

Aspectos tecnológicos e sensoriais do guandu [Cajanus cajan (L.) Millsp.] enlatado em diferentes estádios de maturação

O objetivo deste trabalho é viabilizar a introdução do guandu verde enlatado no mercado brasileiro. Foram realizadas três colheitas de grãos ainda verdes da cultivar IAC Fava Larga obtidos no 44º, 57º e 62º dia após a floração (DAF) e finalmente no 92º DAF, quando os grãos já possuíam cor pardo-amarelada. Para todos os lotes, após otimização das condições de enlatamento, 121ºC durante 5 a 6 minutos, efetuaram-se estudos quanto ao acúmulo de matéria seca, composição dos grãos, cor, textura e aceitação sensorial. Conclui-se, através do acúmulo de matéria seca do grão, que a maturidade fisiológica ocorreu em torno do 62º DAF, quando também apresentou as melhores características para o enlatamento. As cores dos grãos pós-enlatamento, vermelha e amarela (unidades Lab Hunter) apresentaram estáveis até o 62º DAF. A textura foi gradativamente mais firme com o avanço do amadurecimento tendo verificado correlação positiva (r = 0,96) entre medidas de texturas sensorial e instrumental. A aceitação do guandu enlatado cultivar IAC Fava Larga foi apenas regular.

Avaliação do amaciamento de carne bovina de dianteiro (Triceps brachii) pelos métodos de maturação, estimulação elétrica, injeção de ácidos e tenderização mecânica

Vinte e dois músculos Triceps brachii obtidos de 11 bovinos fêmeas com idade 3 e 4 anos, abatidos em abatedouro experimental, foram submetidos à tenderização mecânica, à injeção de ácido acético 0,1M e ácido lático 0,2M, à maturação por 9 e 14 dias, à estimulação elétrica (250V - 60 Hz - 90s), sendo alguns reservados como grupo-controle, sem tratamento. A maturação por 14 dias apresentou 21% de aumento na maciez subjetiva e 12% de diminuição da força de cisalhamento; estes valores são semelhantes aos da estimulação elétrica. Contudo, a injeção com ácidos e a maturação por 9 dias não apresentaram efeito significativo sobre a textura da carne tratada. E embora o valor da força de cisalhamento da carne tenderizada mecanicamente tenha sido o menor entre todos os tratamentos, suspeita-se de superestimação devido ao plano de fraturas criadas por tal processo.Ainda outras análises foram realizadas, como: monitoramento da curva de pH e de valor R; análises de cor; perda de peso por cozimento e devido aos tratamentos; e análises microbiológicas.