RESUMO
The objective of the present study was to quantify the relationships between prepartum nonesterified fatty acid (NEFA) concentrations and the development of subsequent diseases or culling and to identify the optimal thresholds allowing identification of animals at high risk of developing postpartum diseases or being culled. A total of 1,299 Holstein cows from 50 commercial herds located around Saint-Hyacinthe (QC, Canada) were enrolled in this observational study. Blood samples were collected from enrolled cows between 1 and 14 d before calving for serum NEFA quantification. Data concerning postpartum diseases and culling were collected from computerized record systems. The association between prepartum NEFA concentrations and postpartum diseases and culling was quantified using generalized linear mixed models, accounting for parity, season, week of sampling, and herd. Optimal NEFA thresholds were evaluated with receiver operator characteristic curves analysis for all diseases and then confirmed with generalized linear mixed models, considering NEFA as a categorical variable (high or low). Prepartum serum NEFA concentrations were associated with diseases diagnosed during the first 30 d in milk (DIM) and culling within the first 50 DIM. The optimal NEFA threshold associated with diseases was ≥290 µmol/L for retained placenta, ≥300 µmol/L for metritis and abomasal displacement, and ≥280 µmol/L for clinical mastitis and hyperketonemia. The level associated with the occurrence of at least one of these diseases in the first 30 DIM was ≥280 µmol/L, but it was ≥260 µmol/L for culling in the first 50 DIM. No relationship was found between NEFA concentrations and reproductive tract diseases (purulent vaginal discharge or cytological endometritis) or subclinical intramammary infection. Despite the strong relationship between prepartum NEFA concentrations and many diseases, the NEFA optimal threshold accuracy found in our study was low. In conclusion, our results demonstrate a relationship between NEFA concentrations in the 14-d period before calving and the subsequent development of diseases and culling. Prepartum NEFA concentrations thresholds between ≥260 and 300 µmol/L appear to be a strategic choice. However, considering the low accuracy, their use at the cow level should be performed with caution.
Assuntos
Doenças dos Bovinos , Transtornos Puerperais , Gravidez , Feminino , Bovinos , Animais , Ácidos Graxos não Esterificados , Ácido 3-Hidroxibutírico , Doenças dos Bovinos/diagnóstico , Fatores de Risco , Transtornos Puerperais/veterinária , Período Pós-PartoRESUMO
BACKGROUND: Titanium dioxide (TiO2) is broadly used in common consumer goods, including as a food additive (E171 in Europe) for colouring and opacifying properties. The E171 additive contains TiO2 nanoparticles (NPs), part of them being absorbed in the intestine and accumulated in several systemic organs. Exposure to TiO2-NPs in rodents during pregnancy resulted in alteration of placental functions and a materno-foetal transfer of NPs, both with toxic effects on the foetus. However, no human data are available for pregnant women exposed to food-grade TiO2-NPs and their potential transfer to the foetus. In this study, human placentae collected at term from normal pregnancies and meconium (the first stool of newborns) from unpaired mothers/children were analysed using inductively coupled plasma mass spectrometry (ICP-MS) and scanning transmission electron microscopy (STEM) coupled to energy-dispersive X-ray (EDX) spectroscopy for their titanium (Ti) contents and for analysis of TiO2 particle deposition, respectively. Using an ex vivo placenta perfusion model, we also assessed the transplacental passage of food-grade TiO2 particles. RESULTS: By ICP-MS analysis, we evidenced the presence of Ti in all placentae (basal level ranging from 0.01 to 0.48 mg/kg of tissue) and in 50% of the meconium samples (0.02-1.50 mg/kg), suggesting a materno-foetal passage of Ti. STEM-EDX observation of the placental tissues confirmed the presence of TiO2-NPs in addition to iron (Fe), tin (Sn), aluminium (Al) and silicon (Si) as mixed or isolated particle deposits. TiO2 particles, as well as Si, Al, Fe and zinc (Zn) particles were also recovered in the meconium. In placenta perfusion experiments, confocal imaging and SEM-EDX analysis of foetal exudate confirmed a low transfer of food-grade TiO2 particles to the foetal side, which was barely quantifiable by ICP-MS. Diameter measurements showed that 70 to 100% of the TiO2 particles recovered in the foetal exudate were nanosized. CONCLUSIONS: Altogether, these results show a materno-foetal transfer of TiO2 particles during pregnancy, with food-grade TiO2 as a potential source for foetal exposure to NPs. These data emphasize the need for risk assessment of chronic exposure to TiO2-NPs during pregnancy.
Assuntos
Nanopartículas/metabolismo , Placenta/metabolismo , Titânio/metabolismo , Feminino , Humanos , Mecônio/química , Nanopartículas Metálicas/análise , Nanopartículas Metálicas/toxicidade , Modelos Biológicos , Nanopartículas/toxicidade , Perfusão , Gravidez , Titânio/toxicidadeRESUMO
STUDY QUESTION: Do the embryo culture media and plastic materials used during assisted reproductive technology (ART) laboratory procedures expose embryos to bisphenol A (BPA)? SUMMARY ANSWER: BPA was not detected in embryo culture media or protein supplements at concentrations above those encountered in normal patient serum and follicular fluids. WHAT IS KNOWN ALREADY: BPA is strongly suspected of altering the epigenome during mammalian development. Medical devices have been shown to be a source of BPA exposure in adult and neonatal intensive care units. STUDY DESIGN, SIZE, DURATION: An analytical study of ART culture media and plastic labware products was performed under conditions close to routine practice and if BPA was detected, tests were carried out under more stringent conditions. PARTICIPANTS/MATERIALS, SETTING, METHODS: Two single-step embryo culture media, two sequential media and three different protein supplements [a purified human serum albumin (HSA), a synthetic serum substitute, and a recombinant HSA] were tested for BPA. Thirty-three different plastic consumables, used from oocyte collection through to embryo transfer, were tested for their ability to leach BPA into their surrounding environment.BPA concentrations were measured according to a previously described liquid chromatography/mass spectrometry method. This method is linear over the calibration range from 0.5 to 100 ng/ml using a linear model weighted by 1/X² and validated in terms of selectivity, linearity, repeatability, reproducibility and limit of quantification (0.5 ng/ml). MAIN RESULTS AND THE ROLE OF CHANCE: Neither the culture media nor the protein supplements were shown to contain detectable levels of BPA. None of the plastic materials leached BPA into the surrounding medium at levels higher than the upper limit detected previously in serum and follicular fluids in women (about 2 ng/ml). However, the plastic of the three tested strippers used for oocyte denudation/embryo handling did contain BPA. Two of these strippers are made with polycarbonate, a plastic whose synthesis is known to require BPA. LIMITATIONS, REASONS FOR CAUTION: This study is limited to the ART media and materials tested here and using a BPA assay with a limit of quantification at 0.5 ng/ml. A minimum volume was required for testing, and one type of plastic labware could not be tested in conditions identical to those in routine use. WIDER IMPLICATIONS OF THE FINDINGS: Although we demonstrated that some plastic materials used in ART contain BPA, under routine conditions none appear capable of leaching BPA at levels higher than those from maternal internal exposure. However, BPA is strongly suspected of altering the epigenome. Since important epigenetic modifications occur in the early embryonic stage, it is questionable whether plastics that contain BPA, polycarbonate in particular, should be used in the manufacture of plastic consumables for ART procedures. STUDY FUNDING/COMPETING INTERESTS: This work was supported by a grant from the Agence de Biomédecine (AOR 2012) and by a grant from the French Ministry of Health (Clinical Research Hospital Program 2012; no.12-018-0560). The authors declared no competing interest.
Assuntos
Compostos Benzidrílicos/análise , Meios de Cultura/química , Embrião de Mamíferos/efeitos dos fármacos , Fenóis/análise , Cromatografia Líquida , Técnicas de Cultura Embrionária/instrumentação , Exposição Ambiental/análise , Espectrometria de Massas , Plásticos/química , Reprodutibilidade dos Testes , Técnicas de Reprodução Assistida/instrumentaçãoRESUMO
Bovine besnoitiosis is a chronic and debilitating disease observed in many European countries that may cause important economic losses in cattle. The recent widespread of the parasite in Europe had led the European Food Safety Authority to declare bovine besnoitiosis as a re-emerging disease in Europe. Many aspects of the epidemiology of bovine besnoitiosis such as the main routes of transmission are still unclear and need to be further studied. Among the different hypotheses, a sexual transmission has not yet been investigated. Therefore, the aim of this study was to evaluate the presence of Besnoitia besnoiti DNA in the semen of naturally infected bulls by using a highly sensitive method (real-time qPCR). Both pre-sperm and sperm fractions of 40 bulls, including seronegative (n = 11), seropositive subclinically (n = 17), and seropositive clinically (n = 12) infected animals, were collected by electroejaculation and analyzed by real-time qPCR. No B. besnoiti DNA was detected in 27 pre-sperm and 28 sperm fractions of the 40 examined bulls, suggesting that the transmission of B. besnoiti infection by the semen of chronically infected bulls is very unlikely.
Assuntos
Doenças dos Bovinos/parasitologia , Coccidiose/veterinária , DNA de Protozoário/isolamento & purificação , Sarcocystidae/isolamento & purificação , Sêmen/parasitologia , Animais , Bovinos , Doenças dos Bovinos/diagnóstico , Coccidiose/diagnóstico , DNA de Protozoário/genética , Masculino , Reação em Cadeia da Polimerase em Tempo Real/métodos , Reação em Cadeia da Polimerase em Tempo Real/veterinária , Sarcocystidae/genética , Sensibilidade e EspecificidadeRESUMO
The objectives of this herd-level prospective observational cohort study were to describe the proportion of cows with elevated prepartum nonesterified fatty acid concentrations (PropElevNEFA) in dairy herds and to assess the herd-level associations between PropElevNEFA and postpartum diseases, reproductive performance, and culling. From November 2018 to December 2020, a convenience sample of 49 herds was enrolled in this study. Blood sampling (16 to 29 cows per herd) was performed during the week before and during the 2 wk following calving to quantify the concentration of nonesterified fatty acids (NEFA) and ß-hydroxybutyrate acids (BHBA), respectively. Elevated NEFA was defined as ≥280 µmol/L and hyperketonemia as BHBA ≥1.4 mmol/L. Retained placenta, metritis, purulent vaginal discharge, endometritis, and mastitis were diagnosed on-farm following standardized definitions, and success at first artificial insemination (AI) and culling events were recorded. The associations between PropElevNEFA and each individual disease, success at first AI, and culling were evaluated using Bayesian aggregated binomial regression models with weakly informative priors, from the which odds ratio (OR) and the 95% credible intervals (BCI) were obtained. A total of 981 cows were included in the statistical analyses representing 16 to 29 (median = 19) cows per herd. Cows were enrolled in the prepartum period of their first to tenth (median = third) lactation, and 41% of them had an elevated prepartum NEFA concentration. At the herd level, PropElevNEFA varied between 11% and 78% (median = 39%). The odds of metritis (OR = 1.37, 95% BCI = 1.13-1.67) increased for every 10-point increase in PropElevNEFA, whereas the odds of success at first AI decreased (OR = 0.69, 95% BCI = 0.59-0.80). The PropElevNEFA was not associated with the other tested diseases or culling. Our results suggest that the herd-level proportion of cows having elevated prepartum NEFA concentrations is associated with metritis and poor success at first AI in dairy herds.
RESUMO
Due to restriction of the use of BPA, several structural analogues such as BPS and BPF have been proposed for its replacement in many consumer products. This has increased the prevalence of BPS and BPF in urine from tested cohorts. However, these substitutes have similar endocrine disrupting properties to BPA, particularly on reproductive and metabolic functions, which suggests that fetal exposure to these analogues could be of concern for human health. Bisphenols (BPs) are mainly metabolized to glucuronides (BP-Gs), which are considered as inactive but provide a relevant marker of fetal exposure during pregnancy. In most instances, these metabolites are indirectly quantified after hydrolysis and measurement of the corresponding native BPs, which may lead to bias due to spurious BPs contamination during blood collection and/or analyses. We have developed a new method for direct quantification of BP-Gs, which has the advantage of not being affected by errors related to the presence of BPs. First, BP-Gs were extracted from plasma by anion exchange solid phase extraction. They were then labelled with dansyl chloride, using experimentally-optimized incubation conditions, after which the dansyl derivatives were injected into an on-line SPE-UHPLC/MS/MS system. The performance of the method, in terms of sensitivity, precision and accuracy, was evaluated in plasma over a concentration range of 0.05-5 ng/mL. The intra- and inter-day CV% precision were lower than 20% with accuracies ranging from 93% to 115%. The limit of quantification was set at 0.05 ng/mL. The method was then applied to measure BP-Gs in forty-four cord plasma samples. Although no BPF-G was found, BPA-G and BPS-G was determined in almost half of the cord plasma samples with concentration ranges nd-0.089 ng/mL and nd-0.586 ng/mL, respectively.
Assuntos
Sangue Fetal , Espectrometria de Massas em Tandem , Compostos Benzidrílicos , Feminino , Humanos , Fenóis , GravidezRESUMO
Bovine besnoitiosis is a reemerging disease in Europe. The clinically Besnoitia besnoiti infection in bulls is characterized by fever, nasal discharge, and orchitis in the acute phase and by scleroderma in the chronic phase. However, in many bulls, B besnoiti infection remains at a subclinical stage. Bull infertility is an economically relevant consequence of besnoitiosis infection. It is not clear, however, if semen quality returns to normal levels when infected animals have clinically recovered. The aim of this study was to examine the relationship between chronic besnoitiosis and bull sexual function in a region of eastern France, where the disease is reemerging, by comparing semen quality and genital lesions in 11 uninfected, 17 subclinically infected, and 12 clinically infected bulls. The presence of anti-B besnoiti antibodies was detected by Western blot test. Semen was collected by electroejaculation. Bulls clinically infected with B besnoiti showed significantly more genital tract alterations than uninfected or subclinically infected bulls. No relationship was evidenced between besnoitiosis infectious status and semen quality, whereas a significant relationship was noted between genital lesions and semen score. This means that in the absence of moderate to severe genital lesions, chronic bovine besnoitiosis is unlikely to alter semen quality. However, as the presence of infected animals could lead to spread of the disease, culling or separation of clinically infected bulls from the remaining healthy animals is strongly recommended.
Assuntos
Doenças dos Bovinos/parasitologia , Coccidiose/veterinária , Doenças Testiculares/veterinária , Animais , Anticorpos Antiprotozoários/sangue , Estudos de Casos e Controles , Bovinos , Doenças dos Bovinos/sangue , Doença Crônica , Coccidiose/patologia , Masculino , Análise do Sêmen , Doenças Testiculares/parasitologia , Doenças Testiculares/patologiaRESUMO
Analogs of gonadoliberin (GnRH) are widely used in cattle to synchronize estrus and to induce ovulation, as well as for the treatment of ovarian cysts. The aim of this study was to compare the plasma profiles of LH and progesterone and the follicular dynamics in response to the administration of gonadorelin, lecirelin, or buserelin at the dose recommended to induce ovulation. In addition, the biological response to a half dose of lecirelin was assessed. Twelve healthy Holstein female cows were divided into four sequence groups, according to a Latin square design and received the four treatments during the four periods of the study. Before each period, the estrous cycle was synchronized, and on Day 6 or 7 of the ensuing cycle, the time at which it was most likely to have a dominant follicle, 100 µg of gonadorelin, 25 µg of lecirelin, 50 µg of lecirelin, or 10 µg of buserelin was administered to the cows. Blood samples were regularly collected for up to 4 days after the GnRH administrations. The plasma LH response was evaluated for up to 6 hours after administration, and the plasma progesterone response and ovarian follicular dynamics were evaluated for up to 4 days. There was a significantly lower LH release after gonadorelin treatment compared to lecirelin at the doses of 25 or 50 µg and the buserelin treatment. The mean maximal LH concentration after gonadorelin treatment was 2.5 lower than after lecirelin or buserelin treatment and was reached 1 hour earlier. Four days after the GnRH administration (i.e., at Days 10-11 of the estrous cycle), the overall mean increase in plasma progesterone concentration was 70% and did not differ between the treatment groups. The percentage of disappearance of the dominant follicle (84.8% of ovulation and 4.3% of luteinization) after GnRH treatment was high (73%, 82%, 100%, and 100%, for gonadorelin, lecirelin at the doses of 25 and 50 µg, and buserelin, respectively) and did not differ between the GnRH treatments. The follicle disappearance was followed by the emergence of a synchronous follicle wave within 2 days in almost all the heifers. Altogether, our data show that the three GnRH analogs, at the doses indicated for the induction of ovulation or at a half dose for lecirelin, are almost equally effective to induce the disappearance of the dominant follicle at Day 6 to 7 of the estrous cycle.
Assuntos
Busserrelina/administração & dosagem , Bovinos/fisiologia , Hormônio Liberador de Gonadotropina/administração & dosagem , Hormônio Luteinizante/sangue , Oligopeptídeos/administração & dosagem , Progesterona/sangue , Animais , Relação Dose-Resposta a Droga , Sincronização do Estro/métodos , Feminino , Folículo Ovariano/efeitos dos fármacos , Folículo Ovariano/fisiologia , Ovulação/efeitos dos fármacos , Indução da Ovulação/métodos , Indução da Ovulação/veterináriaRESUMO
The aim of this study was to identify the origin of scrapie-induced hypercortisolism. Cortisol and ACTH kinetics and production rate were measured in 14 ewes (6 healthy and 8 scrapie-affected). It was shown that cortisol plasma clearance remained unmodified but that cortisol production rate and plasma concentrations of free cortisol were increased by a factor of 5, whereas the total cortisol plasma concentrations were only doubled. The apparent discrepancy between adrenal secretion rate and the corresponding total cortisol plasma levels was attributable to the scrapie-induced lower corticosteroid-binding globulin (CBG) binding capacity, which altered the ratio of free-to-bound cortisol. The secretion rate of ACTH from diseased ewes was increased by a factor of 1.5, in comparison with healthy ewes, and 4 of the 8 scrapie-affected ewes exhibited a decreased response to a low dexamethasone suppression test. The administration of tetracosactide induced a 2-fold increase in the cortisol production in diseased ewes, compared with that of healthy ewes, but the pituitary sensitivity to ovine CRF was not modified by the prion disease. In conclusion, natural scrapie displays a syndrome of hypercorticism associated with increased ACTH secretion, hyperresponsiveness of the adrenals, and lower CBG binding capacity, which leads to overexposure to CBG-free cortisol.
Assuntos
Hidrocortisona/sangue , Scrapie/sangue , Glândulas Suprarrenais/metabolismo , Hormônio Adrenocorticotrópico/sangue , Algoritmos , Animais , Área Sob a Curva , Encéfalo/patologia , Hormônio Liberador da Corticotropina/sangue , Hormônio Liberador da Corticotropina/farmacologia , Cosintropina/farmacologia , Dexametasona/farmacologia , Feminino , Glucocorticoides/farmacologia , Meia-Vida , Neuro-Hipófise/metabolismo , Scrapie/metabolismo , Scrapie/patologia , OvinosRESUMO
The 24-h pattern of corticoid plasma concentrations was determined in scrapie-affected ewes during the clinical phase of the disease. Twenty one ewes (8 healthy and 13 scrapie-affected ewes) were subjected to 24-h blood sampling sessions. Urine samples were simultaneously obtained during the clinical stage of the disease and in healthy ewes. The scrapie diagnosis was performed by histopathology. Plasma and urinary corticoids were assayed using HPLC methods. Mean plasma and urinary levels of corticoid (cortisol, 20beta-dihydrocortisol cortisone) of scrapie-affected ewes were greater than those observed in healthy ewes. 20Beta-dihydrocortisol appeared to be the main cortisol metabolite in ewes. The intra-individual variations of 20beta-dihydrocortisol plasma concentrations were lower than the corresponding plasma cortisol concentrations due to the dampening effect of the metabolic process on the short term variations of cortisol secretion. This dampening mechanism was amplified in urine, the urinary concentrations integrating cortisol production over the period preceding sampling. For these reasons, 20beta-dihydrocortisol could present a potential interest for a non invasive diagnostic test of transmissible spongiform encephalopathies. The pathophysiological consequences of an excessive exposure to cortisol on development of the neurogenerative process are discussed.
Assuntos
Corticosteroides/sangue , Corticosteroides/urina , Scrapie/metabolismo , Animais , Encéfalo/patologia , Cromatografia Líquida de Alta Pressão , Ritmo Circadiano , Cortisona/sangue , Cortisona/urina , Feminino , Hidrocortisona/análogos & derivados , Hidrocortisona/sangue , Hidrocortisona/urina , Scrapie/diagnóstico , Scrapie/patologia , OvinosRESUMO
We have shown previously that chronic treatment with glucocorticoids enhances both ACTH-induced cAMP production and ACTH- or 8Br-cAMP-induced steroidogenesis of cultured ovine adrenocortical cells. This treatment has been shown to involve an increase in the number of ACTH receptors. The present study aimed to explore the mechanism of this effect of glucocorticoids on ACTH receptors. Ovine adrenocortical cells expressed one major ACTH receptor transcript of 3.6 kb and three minor ones of 4.2, 1.8 and 1.3 kb. Dexamethasone treatment of cultured cells increased the levels of all these transcripts in a time- and dose-dependent manner, with an EC50 of (1.5 +/- 0.6) x 10(-8) M. The mean increase over control with 10(-6) M dexamethasone was 144 +/- 11% (n = 14). This enhancing effect was specific for glucocorticosteroids. The antiglucocorticoid Ru38486 blocked the effect of dexamethasone. Testosterone did not modify, while high concentrations of 17 beta-estradiol decreased, ACTH receptor mRNA levels. Treatment of cells with aminoglutethimide (an inhibitor of steroidogenesis) resulted in a dose-dependent decrease in ACTH receptor mRNA levels, which was prevented by concomitant treatment with dexamethasone. Treatment with ACTH also increased ACTH receptor mRNA levels more than twofold. Addition of aminoglutethimide together with ACTH resulted in a smaller increase than that achieved with ACTH alone. Neither dexamethasone nor ACTH modified ACTH receptor mRNA half-lives. However, these two hormones enhanced the levels of both newly synthesized and total ACTH receptor mRNAs. These results indicate that the positive trophic effect of glucocorticoids on ovine adrenocortical cells involves an enhancement of the transcription rate of the ACTH receptor gene. In addition, they suggest that part of the trophic action of ACTH on ACTH receptors may be mediated by ACTH-induced steroidogenesis.
Assuntos
Córtex Suprarrenal/efeitos dos fármacos , Hormônio Adrenocorticotrópico/farmacologia , Dexametasona/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , RNA Mensageiro/genética , Receptores da Corticotropina/genética , Córtex Suprarrenal/citologia , Córtex Suprarrenal/metabolismo , Animais , Células Cultivadas , RNA Mensageiro/metabolismo , OvinosRESUMO
This study aimed at investigating the possible linkage between natural scrapie and alterations of the somatotropic axis. Scrapie-affected ewes exhibited 2-fold higher mean GH concentrations during both autumn and spring. GH pulse frequencies were higher in scrapie-affected ewes than in control animals (mean+/-S.E.M. number of pulses/24 h: 10.4+/-0.9 and 7.6+/-0.9 for scrapie-affected and control ewes respectively) suggesting the involvement of central mechanisms. GH secretion induced by administration of an alpha(2)-adrenergic agonist, which acts centrally to stimulate GH secretion, was similar between healthy and scrapie-affected ewes (ratios of the area under the curve (AUC) of GH concentration after to the GH AUC before the agonist administration were 3.6+/-1.6 and 4.9+/-1.0 for scrapie-affected and control ewes respectively). Finally, humoral markers and parameters of the metabolic status were determined to test the hypothesis that scrapie-associated alterations of GH secretion could be related to disruption of metabolic homeostasis. Glucose, insulin and urea plasma concentrations were higher in scrapie-affected than in healthy ewes. Neither leptin nor IGF-I levels were affected by scrapie. Total thyroxine (T4) was decreased in scrapie-affected ewes but free T4 and total and free triiodothyronine were not modified. In conclusion, our results showed the existence in scrapie-affected ewes of endocrine and metabolic alterations typical of acute illness proceeding, at least in part, from central mechanisms.
Assuntos
Hormônio do Crescimento/metabolismo , Hipófise/metabolismo , Scrapie/fisiopatologia , Doença Aguda , Agonistas alfa-Adrenérgicos/farmacologia , Animais , Glicemia/análise , Feminino , Hormônio do Crescimento/sangue , Insulina/sangue , Masculino , Hipófise/citologia , Hipófise/efeitos dos fármacos , Scrapie/sangue , Estações do Ano , Taxa Secretória/efeitos dos fármacos , Ovinos , Tiroxina/sangue , Ureia/sangueRESUMO
Naturally scrapie-affected ewes present a syndrome of hypercortisolism as evaluated by measuring total plasma cortisol concentrations. The objective of this study was to investigate the plasma protein binding of cortisol and to evaluate the concentration of the biologically active free fraction of cortisol in scrapie-affected ewes. Corticosteroid binding globulin (CBG) binding parameters were evaluated by equilibrium dialysis in 13 naturally scrapie-affected ewes and nine healthy ewes, during two periods of the clinical evolution of the disease. The hypercortisolism of the scrapie-affected ewes was confirmed by a significant increase of the plasma 20 beta-dihydrocortisol and cortisone concentrations, while total cortisol concentrations, obtained from an isolated sample, did not differ between scrapie-affected and control ewes. The scrapie diagnosis was confirmed by histopathology. The CBG maximal capacity (B(max)) was two times lower in scrapie-affected ewes than in healthy ewes (37+/-32 nM and 73+/-28 nM respectively). The dissociation constant K(d) (8.8+/-3.7 nM and 9.8+/-3.0 nM respectively) and the non-specific constant value of binding to albumin (1.13+/-0.18 and 1.14+/-0.23 respectively) did not differ significantly between diseased and control ewes. The significant increased concentrations of CBG-free cortisol (i.e. both albumin-bound and free cortisol fractions) in scrapie-affected ewes indicates that total plasma cortisol concentration is not an appropriate index of pituitary-adrenocortical hyperactivity. In conclusion, ewes with naturally occurring scrapie display a syndrome of hypercortisolism associated with a lower CBG binding capacity which leads to an overexposure of glucocorticoid-sensitive targets to CBG-free cortisol. The physiopathological consequences of this overexposure on the development of the neurodegenerative process in prion disease are discussed.
Assuntos
Hidrocortisona/metabolismo , Scrapie/metabolismo , Transcortina/metabolismo , Animais , Ligação Proteica , OvinosRESUMO
We have shown previously that a chronic treatment with glucocorticoids enhances cAMP- or ACTH-induced steroidogenesis of cultured ovine adrenocortical cells. This effect appears to involve a greater amount of cholesterol in mitochondria. Hence, the present study aimed to define the role of glucocorticoids in cholesterol metabolism by these cells. 2-day-old cultures were exposed to different hormones or inhibitors (10(-6) M ACTH, 10(-5) M metyrapone) for 28-48 h. At the end of the treatment period, the cells were stimulated for 2 h with 10(-3) M 8Br-cAMP, in the presence of 10(-3) M aminoglutethimide (in order to load mitochondria with cholesterol). Mitochondria were then isolated and incubated without or with 100 microM cholesterol either in the presence or absence of 10(-3) M CaCl2, or with 25 microM 22R-hydroxycholesterol. Mitochondria isolated from dexamethasone-treated cells produced consistently more pregnenolone than mitochondria from control cells, suggesting that at least part of the additional cholesterol present in these mitochondria was available for steroidogenesis. However, similar differences were obtained when mitochondria were incubated in the presence of exogenous cholesterol, both with or without calcium, or in the presence of 22R-hydroxycholesterol. Pregnenolone production under these latter conditions was much higher than when endogenous cholesterol was the only substrate. Conversely, metyrapone treatment of the cells resulted in lower production of pregnenolone from 22R-hydroxycholesterol by their mitochondria. Likewise ACTH treatment enhanced pregnenolone production by isolated mitochondria irrespective of the incubation conditions. These effects of dexamethasone and ACTH were not related to higher amounts of adrenodoxin, adrenodoxin reductase or cytochrome P450scc. These results indicate that exposure of ovine adrenocortical cells to glucocorticoids or ACTH enhances their steroidogenic potency not only by increasing the amount of cholesterol available for steroidogenesis but also by enhancing some step(s) involved in the transformation of cholesterol into pregnenolone.
Assuntos
Córtex Suprarrenal/metabolismo , Colesterol/metabolismo , Dexametasona/farmacologia , Glucocorticoides/farmacologia , Mitocôndrias/metabolismo , 8-Bromo Monofosfato de Adenosina Cíclica/farmacologia , Córtex Suprarrenal/citologia , Corticosteroides/biossíntese , Hormônio Adrenocorticotrópico/farmacologia , Adrenodoxina/biossíntese , Aminoglutetimida/farmacologia , Animais , Cloreto de Cálcio/farmacologia , Células Cultivadas , Colesterol/farmacologia , Inibidores das Enzimas do Citocromo P-450 , Sistema Enzimático do Citocromo P-450/metabolismo , Inibidores Enzimáticos/farmacologia , Ferredoxina-NADP Redutase/biossíntese , Glucocorticoides/antagonistas & inibidores , Hidroxicolesteróis/metabolismo , Hidroxicolesteróis/farmacologia , Metirapona/farmacologia , Mitocôndrias/efeitos dos fármacos , Pregnenolona/biossíntese , RNA Mensageiro/biossíntese , Ovinos , Esteroides/farmacologiaRESUMO
Semen production units compete heavily with each other, so they tend to select and collect bulls at the earliest possible age, even before puberty, in order to reduce the interval between generations. This study is a retrospective analysis of the effect of precocious collection on semen quality in Holstein bulls. The semen parameters of early- and late-maturing bulls collected before and after 410 days of age, respectively, were compared over two periods, 1991-1995 and 1997-1999. These periods were defined in relation to the collection rhythms (three collections of two ejaculates at 15 days interval before 1996 and adaptation of the collection rhythms to individual physiological capacity after 1996) and the collection conditions. The effects of age, precocious collection and the interaction between age and precocious collection on mean semen parameters (volume of the ejaculate, sperm motility, percent of motile spermatozoa per ejaculate, total sperm concentration and mobile sperm concentration) measured on collections 1-6 (n = 358 for 1991-1995 and n = 121 for 1997-1999), 7-12 (n = 255 for 1991-1995 and n = 80 for 1997-1999) and 13-18 (n = 92 for 1991-1995 and n = 36 for 1997-1999) were studied by covariance analysis. The semen quality of bulls collected at the early age differed from that of bulls collected after 410 days of age for the first period when the collection rhythm was intense. No effect of precocious collection was evidenced for the second period, suggesting the importance of individual adaptation of the collection rhythm to sexual maturation in young bulls. Early collections at a semen production unit reduced the time needed to obtain the number of insemination straws required for the progeny-testing program by 40 days. Early sperm collection is, thus, of economic and technical interest in well managed semen production units.
Assuntos
Envelhecimento , Bovinos/fisiologia , Sêmen/fisiologia , Coleta de Tecidos e Órgãos/veterinária , Animais , Inseminação Artificial/veterinária , Masculino , Maturidade Sexual , Motilidade dos Espermatozoides , Coleta de Tecidos e Órgãos/métodosRESUMO
The mean (sd) concentration of plasma 20beta-dihydrocortisol in 126 scrapie-affected sheep was 5-5 (7.0) ng/ml compared with 1.1 (0.7) ng/ml in 52 healthy sheep. The mean (sd) concentration of creatinine in the urine of 93 scrapie-affected sheep was 2.43 (1.56) microg/ml compared with 0.94 (0.86) pg/ml in 49 healthy sheep and 1.10 (0-95) pg/ml in 25 sheep with other diseases. These discriminant analyses carried out on healthy and scrapie-affected sheep showed that plasma 20beta-dihydrocortisol and urinary creatinine were the best predictors of the disease, and classified correctly 98 per cent of healthy sheep and 82 per cent of scrapie-affected sheep.
Assuntos
Creatinina/urina , Hidrocortisona/análogos & derivados , Hidrocortisona/sangue , Scrapie/diagnóstico , Animais , Biomarcadores/sangue , Biomarcadores/urina , Estudos de Casos e Controles , Feminino , Testes Hematológicos/normas , Testes Hematológicos/veterinária , Masculino , Valor Preditivo dos Testes , Gravidez , Scrapie/sangue , Scrapie/urina , Ovinos , Urinálise/normas , Urinálise/veterináriaRESUMO
The widespread human exposure to Bisphenol A (BPA), an endocrine disruptor interfering with developmental processes, raises the question of the risk for human health of BPA fetal exposure. In humans, highly variable BPA concentrations have been reported in the feto-placental compartment. However the human fetal exposure to BPA still remains unclear. The aim of the study was to characterize placental exchanges of BPA and its main metabolite, Bisphenol A-Glucuronide (BPA-G) using the non-recirculating dual human placental perfusion. This high placental bidirectional permeability to the lipid soluble BPA strongly suggests a transport by passive diffusion in both materno-to-fetal and feto-to-maternal direction, leading to a calculated ratio between fetal and maternal free BPA concentrations of about 1. In contrast, BPA-G has limited placental permeability, particularly in the materno-to-fetal direction. Thus the fetal exposure to BPA conjugates could be explained mainly by its limited capacity to extrude BPA-G.
Assuntos
Compostos Benzidrílicos/metabolismo , Disruptores Endócrinos/metabolismo , Glucuronídeos/metabolismo , Fenóis/metabolismo , Placenta/metabolismo , Feminino , Humanos , Técnicas In Vitro , Troca Materno-Fetal , Perfusão , GravidezRESUMO
Bisphenol A (BPA) is a widely used plasticizer that can contaminate food and the wider environment and lead to human exposure. In humans, it is mainly metabolized to bisphenol A-glucuronide (BPA-G) and eliminated in the urine. As BPA causes adverse physiological effects at low doses, it is necessary to document the toxicokinetics of both molecules for risk assessment. Because BPA-G is not available as an analytical standard, it is usually quantified after the assay of BPA, following an enzymatic hydrolysis with ß-glucuronidase. With this approach, two separate assays are required for BPA and BPA-G quantification, which can lead to critical pitfalls in terms of accuracy and analysis time. To overcome this problem, we have developed a new method for the isolation and purification of BPA-G from urine by flash chromatography. Large amounts of BPA-G (1g) were isolated and characterized by mass spectrometry and NMR. This BPA-G is suitable for an use as analytical standard and enabled us to develop a novel method for the simultaneous quantification of BPA and BPA-G in biological matrices by UPLC/MS/MS. It has also been used for in vivo toxicokinetic studies in sheep. The method of quantification was validated according FDA guidelines and used to monitor the time course of plasma and urine concentrations of BPA or BPA-G following their administration. The simultaneous quantification of BPA and BPA-G was compared to the commonly used method for urine and plasma samples. For plasma samples, the results obtained with the direct assay of BPA-G were similar to those obtained by quantification after enzymatic hydrolysis. With urine samples, the simultaneous quantification appeared to be more suitable than the hydrolysis method for the BPA-G determination.
Assuntos
Análise Química do Sangue/métodos , Glucuronídeos/análise , Glucuronídeos/farmacocinética , Fenóis/análise , Fenóis/farmacocinética , Urinálise/métodos , Animais , Compostos Benzidrílicos , Cromatografia Líquida de Alta Pressão , Glucuronídeos/isolamento & purificação , Glucuronídeos/metabolismo , Humanos , Hidrólise , Fenóis/isolamento & purificação , Fenóis/metabolismo , Reprodutibilidade dos Testes , Espectrometria de Massas em Tandem , Fatores de TempoRESUMO
In this study we tested the hypothesis that photoperiod can modulate steroid access to the brain in a seasonal breeder. To this goal, we compared the passage of exogenous progesterone to the brain of female sheep maintained under short (SD) or long (LD) daylengths. In the first experiment, we studied two groups of ovariectomized females maintained under SD or LD, for three artificial cycles, consisting of bearing a subcutaneous oestradiol implant (E2-treated) and an intravaginal device releasing progesterone (CIDR). During the third cycle, the concentrations of progesterone and of its metabolites 5alpha-dihydroprogesterone and 3alpha-hydroxy-5alpha-pregnan-20-one were measured in the preoptic area (POA). The levels of progesterone in the POA were higher in ewes under LD than under SD while the amounts of metabolites were unchanged. In the second experiment, we compared ovariectomized female sheep equipped with a cannula in the third ventricle to sample the cerebrospinal fluid (CSF) under LD vs. SD. After progesterone (1 mg and 10 mg) was injected into the carotid artery, it was only detectable in the cerebrospinal fluid in sheep under LD. In the third experiment, we compared progesterone concentration in plasma and CSF in two groups of SD vs. LD ovariectomized E2-treated ewes for 2 h under CIDR treatment. Despite similar progesterone plasma concentrations, concentration in the CSF was 2.5 times higher in SD than in LD. Our results suggest a physiological modulation of the passage of progesterone to the brain according to the photoperiod.