Placental vascularization in middle and late gestation in the pig.

The aims of this study were to determine the changes in the capillary area density in relation to fetal development, to determine immunoexpression of angiogenic factors and to compare their mRNA expression throughout pig gestation. Samples were collected from the maternal-chorioallantoic interface at days 40, 77, 85 and 114 of pregnancy for immunohistochemistry analysis and the measurement of mRNA expression of VEGFA, ANGPT1, ANGPT2, FGF2 and its receptors KDR, TEK, FGFR1, FGFR2respectively. Morphometric measurement of blood vessels was performed. We found a significant increase in capillary area density throughout gestation (P< 0.05). On the maternal side, at day 77, we observed a significant increase in the number of vessels from small vascular areas (P < 0.05) and the vascular area was significantly higher on day 85 (P < 0.05). On the fetal side, the number of vessels and the vascular area increased between days 40 and 77 (P < 0.05) and between days 77 and 114 (P < 0.05), respectively. Immunohistochemical findings revealed intense VEGFA staining and a trend for increased expression towards the end of gestation (P < 0.05). We also demonstrated a high VEGFA, FGF2, FGFR1, ANGPT1 and ANGPT2mRNA expression at day 77 (P < 0.05). In conclusion, our findings suggest that an active angiogenesis would be present even until late-middle gestation at day 77 of pregnancy with the predominance of angiogenic stimulation by VEGFA/KDR, FGF2/FGFR1 and a balance between ANGPT1 and ANGPT2/TEK. Lay summary: Critical moments occur at different stages of placental formation in pigs, where the expression of angiogenic factors, that is, molecules that stimulate the formation of blood vessels must be adequate to promote their development. This exchange is necessary to cover the increasing nutritional demands of fetuses in continuous development. Determining the changes in the area of capillary density in relation to fetal development and the expression of angiogenic factors throughout pregnancy in pigs could contribute to understanding the causes of fetal loss. Placental samples were obtained at gestational days 40, 77, 85 and 114 (n = 7, 10, 7 and 5, respectively). We found that the capillary area density increases accompanying fetal growth with advancing gestation and an increase in capillary area density in late-middle gestation, around day 77, is due to the expansion in the number of small blood vessels on the maternal side. The present findings suggest that an intense angiogenesis would be present even until late-middle gestation at day 77 of pregnancy, with the predominance of angiogenic stimulation by specific molecules that promote this process.

Capacity of adherence, invasion and intracellular survival of Streptococcus uberis biofilm-forming strains.

AIM: Nine Streptococcus uberis strains with different biofilm-forming profiles in relation to their capacity of adherence and invasion to MAC-T cell lines were examined. Additionally, virulence genes were also linked to adherence and invasion. METHODS AND RESULTS: All S. uberis were able to adhere and invade the cells at different levels. UB56 strain showed the highest percentage of internalization (3.65%) and presented a moderate level of adhesion (4.6 × 106 ). In contrast, UB152, the most adherent strain (8.7 × 106 ) showed a low capacity to internalize (0.65%). Eight strains were able to persist intracellularly over 96 h regardless of their adherence or invasion level. Statistical analysis between biofilm-forming ability and the adhesion capacity showed no significant differences. Presence of virulence genes involved in the adhesion process (gapC, hasABC, lbp, pauA and sua) showed that the strains harboured different genes and seven patterns could be observed. CONCLUSION: Statistical analysis showed no correlation between the virulence gene patterns and the adhesion capacity or the percentage of internalization. Biofilm-forming ability did not influence the invasion capacity. Likewise, adherence and invasion capacity may be strain dependent. SIGNIFICANCE AND IMPACT OF THE STUDY: Findings from this study provide new insights on biofilm and invasion capacity of S. uberis strains. Results could help to design adequate control strategies.

A Saccharomyces cerevisiae RC016-based feed additive reduces liver toxicity, residual aflatoxin B1 levels and positively influences intestinal morphology in broiler chickens fed chronic aflatoxin B1-contaminated diets.

The present study was conducted to investigate the ability of Saccharomyces cerevisiae RC016 (Sc)-based feed additive to reduce liver toxicity, residual aflatoxin B1 (AFB1) levels and influence intestinal structure in broiler chickens fed chronic aflatoxin B1-contaminated diets. A total of 100 one-day-old male commercial line (Ross) broiler chickens were divided into 4 treatments, with 5 pens per treatment and 5 broiler chickens per pen. Birds were randomly assigned to 4 treatments, which were namely treatment 1 (T1), control diet (CD); T2, CD + Sc at 1 g/kg; T3, CD + AFB1 at 100 µg/kg; T4, CD + Sc at 1 g/kg + AFB1 at 100 µg/kg. The liver histopathology of broiler chickens fed diets with AFB1 showed diffused microvacuolar fatty degeneration. The addition of Sc showed normal hepatocytes similar to the control. The small intestine villi from AFB1 group showed atrophy, hyperplasia of goblet cells, prominent inflammatory infiltrate and oedema. In contrast, the small intestine villi from birds that received the yeast plus AFB1 showed an absence of inflammatory infiltrate, and atrophy; moreover, a lower number of goblet cells compared to the groups with AFB1 was observed. The morphometric intestine studies showed that a significant decrease (P < 0.05) in the crypt depth values when Sc was applied to AFB1-contaminated diets. Although the intestinal villus height and apparent adsorption area did not show significant differences (P > 0.05), there was a tendency to improve these parameters. The residual levels of AFB1 in livers were significantly reduced (P < 0.05) in the presence of the yeast. The present work demonstrated that the addition of Sc alone or in combination with AFB1 in the broiler chicken diets had a beneficial effect in counteracting the toxic effects of AFB1 in livers besides improving the histomorphometric parameters and modulating the toxic effect of AFB1 in the intestine.

Effect of prepubertal nutrition on cellular apoptosis and proliferation in at term placenta of Anglo-Nubian goats.

The aim of this study was to evaluate the effect of feed restriction with or without monensin supplementation, followed by a re-feeding period on cellular apoptosis and proliferation in at term placenta of Anglo-Nubian goats. To evaluate the induction of apoptosis through the intrinsic pathway, proteins Bax and Bcl-2 were determinated. The apoptosis was related with the cell proliferation indices through Ki67 determination. The treatments were applied for 250 days and were (a) ad libitum feeding (control; n = 5); (b) restricted feeding at 70% of control (restricted; n = 7); and (c) restricted with monensin supplementation (monensin; n = 7). After treatments, all the animals were fed to support their requirements. After parturition, 27 placentas were gathered. The placental cellular structure was studied by high-resolution light microscopy and transmission electron microscopy; the cellular proliferation was determined by Ki67 index, and Bax and Bcl-2 proteins were localized by immunohistochemical analysis. Differences in cell proliferation through the Ki67 index were found in monensin group placentas. Monensin supplementation stimulated the placental cell proliferation reversing the effect of feed restriction during the peripuberal period. A significant increase of Bcl-2 in placentas of restricted group was found, and it would provide a protective effect on the placental structure. A lack of the Bcl-2 protective effect was observed in control and monensin group placentas, probably meaning that the observed apoptosis would be induced through the intrinsic signalling pathway. A balance between apoptosis and cell proliferation is necessary to maintain tissue homoeostasis during caprine placental development.

Immunohistochemical determination of Ang-1, Ang-2 and Tie-2 in placentas of sows at 30, 60 and 114 days of gestation and validation through a bioinformatic approach.

Angiopoietins (Ang-1, Ang-2) participate in vascular development and placental growth, both bind to Tie-2. This study aimed to determine the localization of angiopoietins in placental development of sows by immunohistochemistry and to validate the gene expression during gestation through a bioinformatic approach. Samples were collected from fifteen maternal-fetal interface from approximately 30 (n = 5), 60 (n = 5) and 114 (n = 5) days of gestation for immunohistochemistry. A bioinformatic approach was performed by re-analysis of public datasets to determine the increase or decrease of genes involved in angiogenesis during pregnancy. There was no significant statistical difference of Ang-1 during gestation, although there was a tendency to increase from mid- to term-gestation (P = 0.7680). A notable decrease of Ang-2 was observed from early- to term-pregnancy (P ≤ 0.05), consistent with the gene expression determined through bioinformatics. Furthermore, there were greater abundances of Tie-2 at both early and at term periods, but lesser abundances at mid-gestation (P ≤ 0.05). The bioinformatics approach indicated that genes related to biological processes such as angiogenesis (i.e., development and morphogenesis of blood vessels) were expressed to a greater extent in early gestation as compared with later in gestation. The Ang-1 gene expression related to cell maturation, response to hypoxia and apoptosis, however, increased as gestation period advanced. In conclusion, angiopoietins may have an important role in the vascular development thus ensuring adequate placental growth in sows. The presence of angiopoietins in the trophoblast suggests a specific role for these pro-angiogenic factors in the tissue formation at the maternal-fetal interface.

Apoptosis and cell proliferation in porcine placental vascularization.

The placenta is a highly vascularized organ, indispensable tothe transfer of nutrients to the growing fetuses. During gestation, there exists an expansion of the placental vascular network through active angiogenesis. The aim of this research was to study cell proliferation and apoptosis through high resolution light microscopy (HRLM) and transmission electron microscopy (TEM) ultrastructure, immunohistochemistry for Ki67and caspase-3, determination of placental vascular area,and TUNEL assay. Crossbred sows placental tissues from approximately 30±2(n=5), 40±2(n=5), 60±2 (n=5), 80±2(n=5), 90±2(n=5) and 114±2(n=5) days of gestation were used. The evaluation of cell proliferation showed the highest%Ki67 values on days 30±2 and 80±2 of pregnancy. Caspase-3 expressed the highest value on day 30±2, while the highest apoptotic indexes were found on days30±2 and 90±2. The placental vascular area was higher on day 80±2 of pregnancy. According to our results, an active vascular cell remodeling by a caspase-3 dependent apoptosis seems to be present in early pregnancy. The increase in the vascular area on day 80±2 would be the result of the intense vascular cell proliferation detected with Ki67. Further studies are needed to understand the complex processes of angiogenesis, cell proliferation and apoptosis that interact in the placenta during porcine gestation.

Actividad sérica del Factor Precoz de Preñez (EPF) durante la gestación en porcinos / Serum Early Pregnancy Factor (EPF) activity during gestation in sows

Para lograr una preñez exitosa es necesario un reconocimiento inmunológico entre la madre y el feto. Tal reconocimiento se realizaría a través de señales como el Factor precoz de preñez (EPF) y de hormonas como la progesterona, que se sintetizan muy tempranamente y poseen actividad inmunosupresora. En este trabajo, se estudió el comportamiento de ambas sustancias durante toda la gestación en porcinos. Se investigó en el suero de 5 cerdas, la actividad de EPF y la concentración de progesterona en estro y en los días 3, 7, 14, 21, 28, 45, 75, 90 y 110 post-servicio. La actividad del EPF mostró un perfil bifásico. Durante el primer tercio de la gestación, en todos los casos el valor máximo de progesterona, apareció 7 días después de los valores máximos de la actividad EPF. Por el contrario, en los dos tercios restantes, los valores máximos de EPF coinciden con los valores máximos de progesterona. De los resultados obtenidos podemos concluir que el EPF sería un buen indicador de fertilización y trazador de viabilidad embrionaria.