Efeito de protocolo hormonal e programa de luz artificial na gestação de éguas receptoras de embrião durante o período de transição de primavera / Effect of hormonal protocol and artificial light program on the pregnancy of embryo recipient mares during spring transition period

O objetivo do estudo foi comparar o efeito das técnicas hormonais e de luz artificial nas éguas receptoras de embrião acíclicas avaliando as taxas de gestação aos 14 e 28 dias durante a fase de transição de primavera. Os 48 animais foram distribuídos aleatoriamente nos grupos: controle (CONT, n=16), éguas cíclicas na fase ovulatória; luz artificial (LUZ, n=16), éguas acíclicas submetidas ao tratamento de luz artificial; e hormônio (HORM, n= 16), éguas acíclicas submetidas ao protocolo hormonal na fase de transição. As éguas do grupo LUZ foram estimuladas por 60 dias com luz artificial durante cinco horas por dia. Nos grupos CONT e LUZ, quando observada a presença de folículo ≥ 35 mm de diâmetro e edema uterino ≥ grau II, foram administrados 1,5 mg de acetato de deslorelina e 1500 UI de hCG para induzir a ovulação. As éguas do grupo HORM foram tratadas com três doses de 1,5 mg de benzoato de estradiol e seguiram os mesmos protocolos dos Grupos CONT e LUZ. Foi avaliada a taxa de gestação por ultrassonografia aos 14 dias e confirmação aos 28 em todos os grupos experimentais. Foi realizada análise descritiva e teste Qui-quadrado (significância de 5%). Taxas de gestação aos 14 e 28 dias foram semelhantes (p>0,05) entre todos os grupos. Os tratamentos HORM e LUZ durante o período de transição inverno-primavera mostraram-se eficazes para atender ao programa de transferência de embrião. Por ser um método mais natural, o protocolo LUZ tem potencial como mais uma ferramenta biotecnológica na reprodução de equinos.

The aim of this study was to compare the effect of hormonal and artificial light techniques on acyclic embryo recipient mares by assessing pregnancy rates at 14 and 28 days during the spring transition period. The 48 animals were randomly assigned to the groups: control (CONT, n = 16), cyclic mares in the ovulatory phase; artificial light (LIGHT, n = 16), acyclic mares subjected to artificial light treatment; and hormone (HORM, n = 16), acyclic mares submitted to hormonal protocol in transition phase. In the LIGHT group, mares were stimulated with artificial light for five hours a day, for 60 days. In CONT and LIGHT groups, when a follicle ≥ 35 mm in diameter and uterine edema ≥ grade II were observed, 1.5 mg of deslorelin acetate and 1500 IU hCG were administered to induce ovulation. In the HORM group, mares were treated with three doses of 1.5 mg of estradiol benzoate and followed the same protocols as the CONT and LIGHT groups. Pregnancy rate was assessed by ultrasound at 14 days and confirmation at 28 days in all experimental groups. Descriptive analysis and chi-square test (5% significance) were performed. Pregnancy rates at 14 and 28 days were similar (p> 0.05) among all groups. The HORM and LIGHT treatments during the winter-spring transition period proved to be effective during the embryo transfer programs. As it is a more natural method, the LIGHT protocol has the potential to be one more biotechnological tool in equine reproduction.

Neurobehavioral and neuroprotector effects of caffeine in animal models

This review aims to analyze and contrast the neurological effects associated with the use of caffeine on neurobehavior and neuroprotection in animal models. Caffeine belongs to the group of methylxanthines that exert a direct effect on adenosine receptors associated with inhibitory or excitatory G proteins, generating modification of cyclic AMP activity and intracellular calcium flow which produces alterations in the modulation system of the neurotransmitters dopamine and glutamate. The regulation of the neurotransmission systems generates protection against the inflammation of the central nervous system, by activation of the microglia and reinforcement of the blood-brain barrier. This drug will also restore cognition or prevent memory loss in Parkinson's or Alzheimer's diseases. It is important to establish new study models in other species to assess whether the behavior of the molecule is similar and to obtain other clinical applications in its behavioral and neuroprotective effects.(AU)

Neurobehavioral and neuroprotector effects of caffeine in animal models

This review aims to analyze and contrast the neurological effects associated with the use of caffeine on neurobehavior and neuroprotection in animal models. Caffeine belongs to the group of methylxanthines that exert a direct effect on adenosine receptors associated with inhibitory or excitatory G proteins, generating modification of cyclic AMP activity and intracellular calcium flow which produces alterations in the modulation system of the neurotransmitters dopamine and glutamate. The regulation of the neurotransmission systems generates protection against the inflammation of the central nervous system, by activation of the microglia and reinforcement of the blood-brain barrier. This drug will also restore cognition or prevent memory loss in Parkinson's or Alzheimer's diseases. It is important to establish new study models in other species to assess whether the behavior of the molecule is similar and to obtain other clinical applications in its behavioral and neuroprotective effects.

Control of oocyte maturation

Oocyte maturation is a complex process involving nuclear and cytoplasmic maturation. The nuclear maturation is a chromosomal segregation and the cytoplasmic maturation involves the reorganization of the cytoplasmic organelles, mRNA transcription and storage of proteins to be used during fertilization and early embryo development. The mechanism of oocyte maturation in vivo and in vitro still are not totally understood. However it is generally accepted that the second messenger cyclic adenosine monophosphate (cAMP) plays a critical role in the maintenance of meiotic blockage of mammalian oocytes. A relative increase in the level of cAMP within the oocyte is essential for maintaining meiosis block, while a decrease in cAMP oocyte concentration allows the resumption of meiosis. The oocyte cAMP concentration is regulated by a balance of two types of enzymes: adenylate cyclase (AC) and phosphodiesterases (PDEs), which are responsible for the synthesis and degradation of cAMP, respectively. After being synthesized by AC in cumulus cells, cAMP are transferred to the oocyte through gap junctions. Thus, specific subtypes PDEs are able to inhibit or attenuate the spontaneous meiotic maturation of oocytes with PDE4 primarily involved in the metabolism of cAMP in granulosa cells and PDE3 in the oocyte. Although the immature oocytes can resume meiosis in vitro, after being removed from antral follicles, cytoplasmic maturation seems to occur asynchronously with nuclear maturation. Therefore, knowledge of the oocyte maturation process is fundamental for the development of methodologies to increase the success of in vitro embryo production and to develop treatments for various forms of infertility. This review will present current knowledge about the maintenance of the oocyte in prophase arrest, and the resumption of meiosis during oocyte maturation, focusing mainly on the changes that take place in the oocyte.

Control of oocyte maturation

Oocyte maturation is a complex process involving nuclear and cytoplasmic maturation. The nuclear maturation is a chromosomal segregation and the cytoplasmic maturation involves the reorganization of the cytoplasmic organelles, mRNA transcription and storage of proteins to be used during fertilization and early embryo development. The mechanism of oocyte maturation in vivo and in vitro still are not totally understood. However it is generally accepted that the second messenger cyclic adenosine monophosphate (cAMP) plays a critical role in the maintenance of meiotic blockage of mammalian oocytes. A relative increase in the level of cAMP within the oocyte is essential for maintaining meiosis block, while a decrease in cAMP oocyte concentration allows the resumption of meiosis. The oocyte cAMP concentration is regulated by a balance of two types of enzymes: adenylate cyclase (AC) and phosphodiesterases (PDEs), which are responsible for the synthesis and degradation of cAMP, respectively. After being synthesized by AC in cumulus cells, cAMP are transferred to the oocyte through gap junctions. Thus, specific subtypes PDEs are able to inhibit or attenuate the spontaneous meiotic maturation of oocytes with PDE4 primarily involved in the metabolism of cAMP in granulosa cells and PDE3 in the oocyte. Although the immature oocytes can resume meiosis in vitro, after being removed from antral follicles, cytoplasmic maturation seems to occur asynchronously with nuclear maturation. Therefore, knowledge of the oocyte maturation process is fundamental for the development of methodologies to increase the success of in vitro embryo production and to develop treatments for various forms of infertility. This review will present current knowledge about the maintenance of the oocyte in prophase arrest, and the resumption of meiosis during oocyte maturation, focusing mainly on the changes that take place in the oocyte. (AU)

Temporal historical observations, rapidly expanding technological tools, and integration of scientific disciplines to enhance reproductive performance of lactating dairy cows: the foster mothers of the human race

Background: Lactating dairy cow of the 21st century is considered to be sub-fertile after intensive selection for milk yield. Production of the dairy cows exemplifies the progress that can be made in the application of technology and holistic dairy cow management to optimize production. The hormonal and metabolic responses, associated with homeorrhetic and homeostatic regulatory responses to partition nutrients for lactation, coupled with intensive management contribute to the reduction in fertility. The numerous postpartum reproductive and metabolic disorders are associated with sub-optimal fertility in the breeding period. Continual advancements have been made in reproductive/lactation physiology, endocrinology, nutrition, herd health and management to improve reproductive herd fertility on commercial dairies. Objective of this presentation is to focus on current technologies and experimental approaches relative to their application in unraveling certain biological windows, which will further impact our ability to enhance reproductive efficiency coupled with increased food production and well-being of both animals and humans. Review: Feeding of omega 6- and omega 3- polyunsaturated fatty acids exert pro- and anti- inflammatory effects on the innate immune system that increases subsequent reproductive performance. Chronic exposure to a GnRH-agonist induced a marked atrophy of the postpartum uterus. Colostrum contains a pleothera of growth factors (lactocrine secretions) that influence uterine developmental programming in the pig, and neonatal exposure to estrogens/progesterone in pigs or cattle alters early programming of the uterus leading to dysfunctional reproductive tract consequences in the adult. Physiological systems to optimize ovarian and uterine function have led to timed insemination fertility programs that have enhanced herd pregnancy rates. Sequencing of the bovine genome has provided an array of new technological approaches to unravel the multi-factorial control systems to support conceptus-placental development throughout gestation and avoid pregnancy failure. A bovine microarray identified genes that were differentially expressed in conceptus and endometrial tissues at day 17 post-LH surge in cyclic and pregnant cows that were lactating or nonlactating. Expression of PAG genes within the conceptus and endometrium of pregnant cows and their association with other genes determined by standard partial correlation analyses infer a possible role of PAG in pregnancy maintenance and implantation by regulation of embryo development, trophoblast cell invasion, immune regulation, and prostaglandin metabolism. The associations detected are suggestive of potential pathways for investigation in early pregnancy at day 17 involving potential direct and indirect effects of PAG 11 produced by the conceptus. Development of microarrays of single nucleotide polymorphisms (SNP) across the bovine genome has led to Genomic Predicted Transmitting Abilities (GPTA) for various production traits including daughter pregnancy rates. More specific physiological reproductive traits have sufficient heritabilities that warrant consideration for selection. Furthermore, current technological advances are identifying candidate "fertility" genes for potential genetic selection, selection for production, health and reproductive traits will be the wave of the future as genomic and bioinformatic tools continue to be expanded and refined. Conclusion: This manuscript targets biological windows and technological advancements to improve reproductive performance of lactating dairy cows. Epidemiological analyses reveal that healthy postpartum lactating dairy cows are indeed fertile. Chronic exposure to a GnRH agonist induced postpartum uterine atrophy warranting additional research as to potential strategies to improve uterine health. Feeding of nutraceuticals such as polyunsaturated omega-6 and omega-3 fatty acids improves postpartum innate immune function and subsequent reproductive performance. Lactocrine secretions in colostrum and neonatal exposure to estrogens and progesterone influence uterine developmental programming related to subsequent reproductive competence. Reproductive management programs that optimize ovarian and uterine function permit a fertile single timed insemination to first and second inseminations. The sequencing of the bovine genome has led to thorough characterizations of the endometrium and conceptus transcriptones in response to key physiological periods such as pregnancy and lactation. Early expression of Pregnancy Associated Glycoprotein (PAG) genes within the conceptus and endometrium of pregnant cows and their association with other genes infer a possible role of PAG in pregnancy maintenance and implantation. The array of SNPs across the bovine genome and specific SNPs within candidate genes related to reproductive processes and fertility will enhance genetic selection for fertility along with production and health associated traits.

Secreção de insulina: interações catiônicas, AMP cíclico e sistema efetor na célula beta / Secreção de insulina: interações catiônicas, AMP cíclico e sistema efetor na célula beta

The review initially deals with cationic rearrangements in the beta cell, as part of the insulin secretion process. It considers the modulator participation of the cyclic AMP. Finally, it focuses microtubules - microfilaments - plasmatic membrane as secretory effector.

A revisão trata, inicialmente, dos rearranjos catiônicos na célula beta, como parte do processo de secreção de insulina. Considera, a seguir, a participação moduladora do AMP cíclico. Finalmente, focaliza microtúbulos - microfilamentos -membrana plasmática, no papel de efetores da secreção.

Secreção de insulina: interações catiônicas, AMP cíclico e sistema efetor na célula beta / Secreção de insulina: interações catiônicas, AMP cíclico e sistema efetor na célula beta

The review initially deals with cationic rearrangements in the beta cell, as part of the insulin secretion process. It considers the modulator participation of the cyclic AMP. Finally, it focuses microtubules - microfilaments - plasmatic membrane as secretory effector. 

A revisão trata, inicialmente, dos rearranjos catiônicos na célula beta, como parte do processo de secreção de insulina. Considera, a seguir, a participação moduladora do AMP cíclico. Finalmente, focaliza microtúbulos - microfilamentos -membrana plasmática, no papel de efetores da secreção.  

Validation of methodologies for the study of horm one-sensitive lipase in lactating cows adipose tissue / Validação de metodologias para o estudo da lipase sensível a hormônio em vacas em lactação

The objectives of this study were: a) to validate techniques to measure HSL in adipose tissue from lactating cows: and b) to measure activation of HSL by second messengers responsible for HSL phosphorylation. Radioactive labelled trioleine (3H-trioleina) was used as a substrate. The emulsion was prepared by sonication in a buffer solution containing 0.1M phosphate, pH= 7.0, which minimized interference of lipoprotein lipase in the assay. Utilization of a mixture of 25mg/ml de phosphatidil-choline and 15mg)ml phosphatidil-ethanolamine as emulsifiers produced the higher HSL activities. Enzyme was obtained by homogeneization, centrifugation at 90,000xg (50 min) and precipitation at pH= 5.2. Linearity of the assay was demonstrated for changes in amount of enzyme and reaction time. HSL was activated in vitro with cyclic dibutiril AMP (dbcAMP), ATP, protein kinase A and MgCl2, and these are the first published results in the literature for ruminants. Concentrations of 2mM ATP and 3 M dbcAMP were necessary to maximize activation of HSL. Kinectic studies of HSL demonstrated that the Km for the bovine enzyme was 0.3mM of trioleina. Activation of HSL by dbcAMP, determined in various concentrations of substrate, was proportionally higher in concentrations near Km, suggesting that activation alters the interface enzyme-lipid droplet, and not the turnover number. In conclusion, the methodo

Este trabalho objetivou: a) desenvolver técnicas para mensuração da HSL(Lipase sensível a hormônio) em tecido adiposo de vacas em lactação;e b) medir a ativação da HSL por segundos mensageiros responsáveis pela fosforilação da HSL Utilizaram-se como substrato emulsões de trioleina radioativa (3H-trioleina). A emulsão foi preparada por sonicação em solução 0,1M de fosfato, pH= 7,0. A utilização da mistura de 2,5mg/ml de fosfatidilcolina e 1,5mg/ml de fosfatidiletanolamina como emulsificante proporcionou as maiores atividades. Obteve-se a enzima por homogeneização,centrifugação a 90.000xg, por 50 min e precipitação a pH= 5,2. Demonstrou-se linearidade da atividade em função da quantidade de enzima e tempo de reação. Obteve-se ativação da HSL in vitro com dibutiril AMP cíclico (dbcAMP), ATP, proteína quinase-A e MgCl2, sendo estes resultados os primeiros publicados na literatura, com bovinos. Para tecido adiposo de bovinos foram necessárias concentrações de 2mM de ATP e 3 M de dbcAMP para maximizar a ativação da HSL. Estudos da cinética da HSL demonstraram que o Km da enzima bovina foi de 0,3 mM de trioleina. A ativação da HSL por dbcAMP, determinada em diversas concentrações de substrato, foi proporcionalmente maior em concentrações próximas ao Km, sugerindo que a ativação altera a interface enzima-gota de lipídeo. A metodologia parece adequada para medir a atividade da HSL e

Validation of methodologies for the study of horm one-sensitive lipase in lactating cows adipose tissue / Validação de metodologias para o estudo da lipase sensível a hormônio em vacas em lactação

The objectives of this study were: a) to validate techniques to measure HSL in adipose tissue from lactating cows: and b) to measure activation of HSL by second messengers responsible for HSL phosphorylation. Radioactive labelled trioleine (3H-trioleina) was used as a substrate. The emulsion was prepared by sonication in a buffer solution containing 0.1M phosphate, pH= 7.0, which minimized interference of lipoprotein lipase in the assay. Utilization of a mixture of 25mg/ml de phosphatidil-choline and 15mg)ml phosphatidil-ethanolamine as emulsifiers produced the higher HSL activities. Enzyme was obtained by homogeneization, centrifugation at 90,000xg (50 min) and precipitation at pH= 5.2. Linearity of the assay was demonstrated for changes in amount of enzyme and reaction time. HSL was activated in vitro with cyclic dibutiril AMP (dbcAMP), ATP, protein kinase A and MgCl2, and these are the first published results in the literature for ruminants. Concentrations of 2mM ATP and 3 M dbcAMP were necessary to maximize activation of HSL. Kinectic studies of HSL demonstrated that the Km for the bovine enzyme was 0.3mM of trioleina. Activation of HSL by dbcAMP, determined in various concentrations of substrate, was proportionally higher in concentrations near Km, suggesting that activation alters the interface enzyme-lipid droplet, and not the turnover number. In conclusion, the methodo

Este trabalho objetivou: a) desenvolver técnicas para mensuração da HSL(Lipase sensível a hormônio) em tecido adiposo de vacas em lactação;e b) medir a ativação da HSL por segundos mensageiros responsáveis pela fosforilação da HSL Utilizaram-se como substrato emulsões de trioleina radioativa (3H-trioleina). A emulsão foi preparada por sonicação em solução 0,1M de fosfato, pH= 7,0. A utilização da mistura de 2,5mg/ml de fosfatidilcolina e 1,5mg/ml de fosfatidiletanolamina como emulsificante proporcionou as maiores atividades. Obteve-se a enzima por homogeneização,centrifugação a 90.000xg, por 50 min e precipitação a pH= 5,2. Demonstrou-se linearidade da atividade em função da quantidade de enzima e tempo de reação. Obteve-se ativação da HSL in vitro com dibutiril AMP cíclico (dbcAMP), ATP, proteína quinase-A e MgCl2, sendo estes resultados os primeiros publicados na literatura, com bovinos. Para tecido adiposo de bovinos foram necessárias concentrações de 2mM de ATP e 3 M de dbcAMP para maximizar a ativação da HSL. Estudos da cinética da HSL demonstraram que o Km da enzima bovina foi de 0,3 mM de trioleina. A ativação da HSL por dbcAMP, determinada em diversas concentrações de substrato, foi proporcionalmente maior em concentrações próximas ao Km, sugerindo que a ativação altera a interface enzima-gota de lipídeo. A metodologia parece adequada para medir a atividade da HSL e

Involvement of G proteins and cAMP in the production of chitinolytic enzymes by Trichoderma harzianum

The effect of G protein modulators and cyclic AMP (cAMP) on N-acetylglucosaminidase (NAGase) production was investigated during 84 h of growth of a Trichoderma harzianum strain in chitin-containing medium. Caffeine (5 mM), N6--2'-O-dibutyryladenosine 3'5'-cyclic monophosphate sodium salt (dBcAMP) (1 mM) and 3-isobutyl-1-methylxanthine (IBMX) (2 mM) decreased extracellular NAGase activity by 80%, 77% and 37%, respectively. AlCl3/KF (100 µM/10 mM and 200 µM/ 20 mM) decreased the activity by 85% and 95%, respectively. Cholera (10 µ/mL) and pertussis (20 µ/mL) toxins also affected NAGase activity, causing a decrease of approximately 75%. Upon all treatments, protein bands of approximately 73 kDa, 68 kDa and 45 kDa had their signals diminished whilst a 50 kDa band was enhanced only by treatment with cholera and pertussis toxins. N-terminal sequencing analysis identified the 73 kDa and 68 kDa proteins as being T. harzianum NAGase in two different truncated forms whereas the 45 kDa band comprised a T. harzianum endochitinase. The 50 kDa protein showed sequence similarity to Coriolus vesicolor cellobiohydrolase. The above results suggest that a signaling pathway comprising G-proteins, adenylate cyclase and cAMP may be involved in the synthesis of T. harzianum chitinases.

O efeito de cAMP e de moduladores de proteínas G sobre a produção de N-acetilglicosaminidase (NAGase) foi investigado durante o crescimento de Trichoderma harzianum em meio contendo quitina. Cafeína (5 mM), dBcAMP (1mM) e IBMX (2 mM) provocaram diminuições na atividade extracelular de NAGase em 80%, 77% e 37%, respectivamente. Por outro lado, a presença de AlCl3/KF nas concentrações de 100 µM/10 mM e 200 µM/ 20 mM causou decréscimo na atividade em 85% e 95%, respectivamente. A toxina do cólera (10 µ/mL) e a toxina pertussis (20 µ/mL) também afetaram a atividade de NAGase, causando um decréscimo de aproximadamente 75%. Análises eletroforéticas mostraram que todos os tratamentos citados causaram diminuição no sinal de bandas correspondendo a polipeptídios de 73 kDa, 68 kDa e 45 kDa, enquanto uma banda de 50 kDa foi intensificada apenas com tratamento com as toxinas do cólera e pertussis. Análises de sequenciamento N-terminal permitiram a identificação das proteínas de 73 kDa e 68 kDa como sendo NAGase de T. harzianum em duas formas diferentemente processadas enquanto a banda de 45 kDa correspondeu a uma endoquitinase de T. harzianum. A proteína de 50 kDa mostrou similaridade de sequência com uma celobiohidrolase de Coriolus vesicolor. Os resultados sugerem que uma via de sinalização composta por proteínas G, adenilato ciclase e cAMP possa estar envolvida na produção de quitinases T. harzianum.