Vascular characterization and morphogenesis in porcine placenta at day 40 of gestation.

In porcine placenta, abnormal development of the placental vasculature leads to placental insufficiency. The aim of this study was to determine the mRNA expression of angiogenic growth factors and to determine the vascular characteristics in placenta at day 40 of pig gestation. Samples were collected from maternal-chorioallantoic interface (n = 21) for the measurement of mRNA expression of VEGFA, ANGPT1, ANGPT2, FGF2 and its receptors KDR, TEK, FGFR1IIIc, FGFR2IIIb respectively, and for immunohistochemistry analysis of CD31 and VEGFA. Immunohistochemical analysis of CD31 and VEGFA, morphometric measurement of blood vessels, high-resolution light microscopy and transmission electron microscopy were performed. Capillary area density, number of blood vessels and capillary area were significantly higher on the maternal side than on the fetal side (p < .05). The ultrastructural finding of blood vessels demonstrates close contact with the trophoblastic epithelium. The relative mRNA expression of VEGFA and its receptor KDR was higher compared with the other angiogenic genes. In conclusion, a high mRNA expression of VEGFA and its receptor KDR added to the immunohistochemical results suggest a potential role of these genes in this pathway associated with an increase in the density of the capillary area on the maternal side and a reduction in the hemotrophic diffusion distance at the interface for nutrient exchange.

Morphometric study of the porcine placental vascularization.

The early development in mammals is characterized by the contribution of nutrients from the maternal tissues through the placenta, which is in apposition with foetal membranes and the endometrium, allowing the physiological interchange between the embryos/foetuses and the mother. The aim of this work was to study the number of placental blood vessels and their vascular area through morphometric analyses and the haemotrophic diffusion distance in porcine placental tissues from early gestations, intermediates gestations, advanced gestations and term gestations. For those purposes, morphometric measurements, blood vessel quantification, high-resolution light microscopy and transmission electron microscopy were performed. The implementation of the high-resolution light microscopy allowed studying the placental vascular and tissue histoarchitecture with higher definition and resolution than using a conventional light microscopy. We highlight the close location of the subepithelial capillaries to the maternal/foetal interface as pregnancy progresses. We found statistically significant evidence to state that the area of blood vessels is dependent on the gestation period. In advanced gestations, the presence of numerous small blood vessels and its near location to foetal/maternal interface agree with the great remodelling reported in our previous studies. In conclusion, in gilts, given the type of non-invasive epithelial placentation, the new blood vessels generation and of haemotrophic diffusion distance reduction, determined in this report, assure the maternal/foetal haemotrophic exchange efficiency during gestation.

Porcine placental immunoexpression of vascular endothelial growth factor, placenta growth factor, Flt-1 and Flk-1.

Porcine embryo mortalities cause economic losses. Development of the placental vascular bed is required for successful gestation and postnatal survival. We studied the temporal and spatial distributions of vascular endothelial growth factor (VEGF), placenta growth factor (PlGF) and their receptors, Flt-1 and Flk-1. We used crossbred swine placental tissues from 30, 60, 80, 90 and 114 (term) days of gestation. Both VEGF and PlGF were present during gestation. At early pregnancy and at term, VEGF probably acts through Flt-1; during intermediate periods, its function is mediated by Flk-1. By day 90, factors other than members of VEGF family appear to be involved.

Cellular remodelling by apoptosis during porcine placentation.

The mechanisms that regulate the apoptosis are essential to the normal development and maintenance of homoeostasis and play an important role in placental development in mammals. During porcine pregnancy, there must be a proper cellular remodelling to achieve a normal gestational development. Knowledge of pig physiology during pregnancy will explore options to increase the productivity of this species of high economical value. The purpose of this work was to study the cell morphology and apoptosis of porcine placentas from early, mid and late pregnancy. For that purpose, high-resolution light microscopy and transmission electron microscopy were performed to the study of cell morphology. TUNEL, the apoptosis index (IAp) and the expression of c-FLIP through immunohistochemistry technique were used to the study of apoptosis. High-resolution light microscopy and transmission electron microscopy confirmed the presence of placental cells with ultrastructural apoptotic features. Apoptotic nuclei were detected by TUNEL in different placental structures and phagocytes containing apoptotic bodies. The IAp in villi was 9.34% at early, 0.82% at mid and 23.85% at late pregnancy. Statistically significant differences were found between periods (p < 0.05). In previous studies, we determined a differential induction of the apoptotic routes in the placental villi in agreement with the gestational period. A co-expression of receptors and mitochondrial proteins in placental connective tissue was detected, but the immunolocalization of c-FLIP would indicate an endogenous inhibition of the extrinsic pathway. In conclusion, in swine there exists differential activation of inducing apoptotic pathways in different placental structures according to the gestational period.

Adsorption of ochratoxin A and zearalenone by potential probiotic Saccharomyces cerevisiae strains and its relation with cell wall thickness.

AIMS: To examine Saccharomyces cerevisae strains with previously reported beneficial properties and aflatoxin B1 binding capacity, for their ability to remove ochratoxin A (OTA) and zearalenone (ZEA) and to study the relation between cell wall thickness and detoxificant ability of yeast strains. METHODS AND RESULTS: A mycotoxin binding assay at different toxin concentrations and the effect of gastrointestinal conditions on mycotoxin binding were evaluated. Ultrastructural studies of yeast cells were carried out with transmission electronic microscopy. All tested strains were capable of removing OTA and ZEA. Saccharomyces cerevisiae RC012 and RC016 showed the highest OTA removal percentage, whereas RC009 and RC012 strains showed the highest ZEA removal percentages. The cell diameter/cell wall thickness relation showed a correlation between cell wall amount and mycotoxin removal ability. After exposure to gastrointestinal conditions, a significant increase in mycotoxin binding was observed. CONCLUSIONS: All tested Saccharomyces cerevisiae strains were able to remove OTA and ZEA, and physical adsorption would be the main mechanism involved in ochratoxin A and ZEA removal. Gastrointestinal conditions would enhance adsorption and not decrease mycotoxin-adsorbent interactions. SIGNIFICANCE AND IMPACT OF THE STUDY: Live strains with mycotoxin binding ability and beneficial properties are potential probiotics that could be included in animal feed. Previous and present results suggest that the RC008 and RC016 strains are very promising candidates for functional feed product development.

Effect of feed restriction and realimentation with monensin supplementation on placental structure and ultrastructure in anglo-nubian goats.

The aim of this study was to evaluate the effect of feed restriction followed by a realimentation with monensin supplementation on morphological, ultrastructural, and apoptotic characteristics in the term placenta of Anglo-Nubian does. Treatments were a control group (C = 5), a group fed at 0.70 of that consumed by controls (R = 7), and the same as R with monensin (M = 7). After parturition, 27 placentas were gathered, C: 7, M: 10, and R: 10. No differences were detected between treatments in relation to morphological and ultrastructural analysis. The greatest values of binucleate cells were detected in placentas from R, and it could be due to the need to compensate and satisfy nutritional differences of restriction. We detected the highest apoptotic index in R as a consequence of nutritional treatment. We describe for the first time the structural and ultrastructural morphology and remodeling by apoptosis of Anglo-Nubian placenta at term of goats subjected to nutritional restriction during peripubertal period and the use of monensin as a growth promoter.

Porcine uterine and placental extracellular matrix molecules throughout pregnancy / Moléculas de la matríz extracelular placentaria y uterina durante la preñez porcina

The molecules that constitute the extracellular matrix are important in several functions related to tissue support and cell-cell, cell-extracellular matrix interaction. Among the macromolecules that constitute the mentioned matrix we find osteopontin, fibrinogen and collagen. The present study was undertaken to analyze the rol of osteopontin, fibrinogen and collagen in uterine-placental interface during normal porcine gestation. Uterine and placental tissues from crossbred gilts of 30 (n=5), 60 (n=5), 70 (n=5) and 114 (at term, n=5) days of gestation were used. Macroscopic analysis of the embryos/fetuses allowed us to determine their gestational age by means of the crown-rump lenght. Haematoxylin-Eosin and Masson's Trichrome dyes along with light microscopy were used to structure analysis of every selected period of gestation. A spacial and temporal study of osteopontin and fibrinogen was performed through immunohistochemical technique. Determination of collagen fibers was carried out through Picrosirius red technique and polarizing microscopy. Results were expressed as semi-quantitative. Higher expression of osteopontin was observed at early periods of gestation, mainly in uterine and placental villi, endometrial gland epithelium and histotroph. Fibrinogen expressed abundantly in fetal mesenchyme in every period analyzed and in fetal and maternal vessels at Day 70. Negative expression of collagen fibers was observed in villi, however increasing expression of thick fibers throughout pregnancy was detected in uterine stroma and myometrium. These results confirm the importance of osteopontin, fibrinogen and collagen in the support of uterine and placental structures and in the suitable maintenance of pregnancy.

Las moléculas que constituyen la matriz extracelular son importantes en varias funciones relacionadas con el soporte del tejido y la interacción célula-célula, célula-matriz extracelular. Entre las macromoléculas que constituyen la matriz mencionada se encuentra la osteopontina, el fibrinógeno y el colágeno. Este estudio se realizó para analizar el rol de la osteopontina, el fibrinógeno y el colágeno en la interface útero-placentaria durante la gestación porcina normal. Tejidos uterinos y la placentarios de hembras porcinas cruzadas de 30 (n=5), 60 (n=5), 70 (n=5) y 114 (a término, n=5) días de gestación fueron utilizados. El análisis macroscópico de los embriones/fetos nos permitió determinar la edad gestacional por medio de la longitud cráneo-rabadilla. Tinciones de Hematoxilina-Eosina y Tricrómico de Masson con microscopía de luz se utilizó para estructurar el análisis de cada periodo de tiempo seleccionado de la gestación. Un estudio espacial y temporal de la osteopontina y el fibrinógeno se realizó mediante técnicas de inmunohistoquímica. La determinación de fibras colágenas se llevó a cabo a través de la técnica Picrosirius rojo por microscopia de polarización. Los resultados se expresaron como semi-cuantitativos. La expresión de osteopontina se observó en los primeros períodos de gestación, principalmente en las vellosidades del útero y la placenta, epitelio de las glándulas endometriales e histotrofos. El fibrinógeno se expresa abundantemente en mesénquima fetal en todos los períodos analizados y en los vasos fetales y maternos el día 70. Una expresión negativa de fibras colágenas se observó en las vellosidades, sin embargo, un aumento de expresión de las fibras gruesas durante la gestación se detectó en el estroma uterino y el miometrio. Estos resultados confirman la importancia de la osteopontina, fibrinógeno y colágeno en el soporte de las estructuras del útero y placenta, así como el mantenimiento adecuado durante la gestación.

Effect of low levels of aflatoxin B1 on performance, biochemical parameters, and aflatoxin B1 in broiler liver tissues in the presence of monensin and sodium bentonite.

Aflatoxins (AF) are a major problem in broiler production and are significant economic and public health burdens worldwide. A commercial sodium bentonite (Na-B) adsorbent was used to prevent the effect of AF [50 µg of aflatoxin B1 (AFB1)/kg of feed] in broiler productivity, biochemical parameters, macroscopic and microscopic liver changes, and AFB1 liver residues. The influence of Na-B (0.3%) and monensin (MON, 100 mg/kg), alone or in combination, was investigated in depth. The dietary treatments were as follows: treatment (T) 1: basal diet (B); T2: B + MON; T3: B + Na-B; T4: B + Na-B + MON; T5: B + AFB1; T6: B + AFB1 + Na-B + MON; T7: B + AFB1 + MON; T8: B + AFB1 + Na-B. Birds were fed dietary treatments for 28 d (d 18 to 46). No significant differences (P < 0.05) were observed among treatments with respect to broiler performance, biochemical parameters, or relative liver weights. With the exception of T8, all livers showed histopathological alterations, with accumulation of fat vacuoles. The normal appearance of livers from T8 showed the protective effect of Na-B against aflatoxicosis. The residual AFB1 levels in livers from T5 to T8 ranged from 0.2 to 1.0 ng/g and were higher in livers from T6 (P < 0.05). Results of this study indicate a competition between AFB1 and MON for adsorption sites on Na-B when feed contains low levels of the toxin, indicating a nonselective adsorption capacity of this particular Na-B. In addition, significant levels of AFB1 in livers indicate that this determination is an important technique not only for diagnosis of aflatoxicosis in broilers, but also for quality control of avian products.

Expression of death cellular receptors FAS/CD95 and DR4 during porcine placentation / Expresión de los receptores de muerte celular FAS/CD95 y DR4 durante la placentación porcina

Apoptosis is a permanent and dynamic physiological process by which an organism eliminates the undesirable cells without causing an inflammatory response. The objective of this work was to study the expression of FAS, DR4 and other members of the TNF-R1 superfamily extrinsic route apoptotic receptors the DNA fragmentation and the cellular apoptosis in placental samples at the early, mid and late pregnancy on +/- 30, +/- 55 and +/- 114 gestational days, respectively. We used placental histological sections of samples fixed in buffered saline formaldehyde. Immunohistochemical techniques were performed to detect the apoptotic receptors, whereas the DNA fragmentation was detected by TUNEL reaction and apoptotic cellular ultrastructure was detected by TEM conventional techniques. Apoptosis related receptors were immunolocalized in the early pig gestation and correlated with apoptosis, suggesting a role in the cellular remodelling of the placenta. At gestation day 55, apoptosis might be correlated to FAS route, but not by DR4-mediating pathway. At the end of gestation, increased apoptosis and both receptors markers were detected showing cellular death due to the extrinsic route through FAS and DR4 receptors. In conclusion, the immunolocalization of FAS and TNF R-1 receptors along the pig placental development correlates with TUNEL reaction and with apoptotic ultrastructure observed by TEM and seems to occur through different pathways along gestation.

La apoptosis es un proceso fisiológico, dinámico y permanente a través del cual un organismo elimina células indeseables sin provocar una respuesta inflamatoria. El objetivo del presente trabajo fue estudiar la expresión de los receptores de la vía extrínseca de apoptosis, FAS, DR4 y otros miembros de la superfamilia TNF-R1, la fragmentación del ADN y la apoptosis celular a través de TEM, en muestras placentarias del inicio, la mitad y el final de la gestación, hacia el día +/- 30, +/- 55 y +/- 114 de preñez, respectivamente. Se realizaron cortes histológicos de las muestras placentarias fijadas en formol tamponado. Para la detección de los receptores de apoptosis se realizaron técnicas inmunohistoquímicas, para el estudio de la fragmentación del ADN se utilizó el ensayo TUNEL y para el análisis de la ultraestructura celular apoptótica la técnica convencional de TEM. La inmunolocalización de los receptores de muerte celular al inicio de la preñez porcina sugiere el rol de la apoptosis en la remodelación celular placentaria. Hacia el día 55 de preñez, la apoptosis detectada ocurriría únicamente a través de la vía del receptor FAS, no del receptor DR4. Al final de la gestación, se detectó un incremento de la apoptosis y la expresión de ambos receptores, indicando que la muerte celular a través de la vía de señalización extrínseca estaría inducida por los receptores FAS y DR4. En conclusión, la inmunolocalización de los receptores FAS y otros miembros del TNF-R1, los resultados de TUNEL y la ultraestructura celular apoptótica observada en la placentación porcina, indican que la apoptosis detectada ocurre por diferentes vías de inducción a lo largo de la gestación.