Detection of nonpregnant cows and potential embryo losses by color Doppler ultrasound and interferon-stimulated gene expression in grazing dairy cows

Many studies have been conducted to estimate pregnancy losses between 19 and 34 d after artificial insemination (AI) in dairy cows managed under confinement-based systems, but few studies have examined embryo mortality during this interval in dairy cows managed under gazing systems. The objectives of this prospective cohort study were (1) to assess the diagnostic value of the corpus luteum (CL) blood perfusion (BP) evaluation by Doppler ultrasound (US) to detect nonpregnant cows at 19 to 20 d post-AI, and (2) to assess the rate of potential embryo mortality between 19 to 34 d post-AI. The CL-BP of all cows included in the study (n = 131) was examined on farm by power and color mode of Doppler US and later using an image processing software by a second evaluator. The endometrium thickness and echotexture were evaluated by B-mode US at the same visit to assess if the nonpregnancy diagnosis could be improved at 19 to 20 d post-AI by this additional diagnostic tool. Blood samples were obtained at 19 to 20 d post-AI for progesterone (P4) measurement by chemiluminescence and to determine the mRNA expression of ISG by real-time PCR. Pregnancy diagnosis based on embryo visualization was performed at 33 to 34 d post-AI by US B-mode. In parallel interpretation, ISG15 and MX2 mRNA expression in leukocytes [sensitivity (Se), 100%] were regarded as suitable biomarkers for early pregnancy and were selected for molecular characterization of pregnancy at 19 to 20 d post-AI. At 19 to 20 d post-AI, 61.1% of the cows had positive CL-BP by Doppler US (Se, 98.0%), 62.7% had ISG mRNA expression in leukocytes over the cutoff point (Se, 95.7%), and 50.8% were positive, based on the combination of ISG mRNA expression, CL-BP by Doppler US, and P4 concentration (Se, 100%), and were considered as possible pregnant. At 33 to 34 d, the pregnancy rate was 37.4% diagnosed by the B-mode US. Based on the expression of the selected biomarkers in cows with active CL, we found that 28.1% of the cows could have potentially lost their pregnancy between 19 and 34 d post-AI. The Doppler US color mode showed similar accuracy and a higher negative predictive value than the genes selected as biomarkers. The additional B-mode ultrasound evaluation of the uterine stratum vasculare and the endometrium thickness improved the diagnostic accuracy. Therefore, assessing the CL-BP by Doppler US allowed early detection of nonpregnant cows at 19 to 20 d post-AI. The combination of early CL-BP by Doppler US (d 19 to 20) with early embryo detection by B-mode US (d 33–34) could be used to facilitate earlier rebreeding of dairy cows

In vivo imaging and quantitative analysis of leukocyte directional migration and polarization in inflamed tissue

Directional migration of transmigrated leukocytes to the site of injury is a central event in the inflammatory response. Here, we present an in vivo chemotaxis assay enabling the visualization and quantitative analysis of subtype-specific directional motility and polarization of leukocytes in their natural 3D microenvironment. Our technique comprises the combination of i) semi-automated in situ microinjection of chemoattractants or bacteria as local chemotactic stimulus, ii) in vivo near-infrared reflected-light oblique transillumination (RLOT) microscopy for the visualization of leukocyte motility and morphology, and iii) in vivo fluorescence microscopy for the visualization of different leukocyte subpopulations or fluorescence-labeled bacteria. Leukocyte motility parameters are quantified off-line in digitized video sequences using computer-assisted single cell tracking. Here, we show that perivenular microinjection of chemoattractants [macrophage inflammatory protein-1alpha (MIP-1alpha/Ccl3), platelet-activating factor (PAF)] or E. coli into the murine cremaster muscle induces target-oriented intravascular adhesion and transmigration as well as polarization and directional interstitial migration of leukocytes towards the locally administered stimuli. Moreover, we describe a crucial role of Rho kinase for the regulation of directional motility and polarization of transmigrated leukocytes in vivo. Finally, combining in vivo RLOT and fluorescence microscopy in Cx3CR1(gfp/gfp) mice (mice exhibiting green fluorescent protein-labeled monocytes), we are able to demonstrate differences in the migratory behavior of monocytes and neutrophils.Taken together, we propose a novel approach for investigating the mechanisms and spatiotemporal dynamics of subtype-specific motility and polarization of leukocytes during their directional interstitial migration in vivo

Diagnosis of embryonic loss and very early detection of non-pregnant cows by Doppler ultrasound Summary

Los productores lecheros enfrentan grandes desafíos que amenazan los resultados económicos del sistema, siendo la eficiencia reproductiva uno de los principales factores para tener en cuenta. Para alcanzar el objetivo de un parto por año/vaca y mantener así la rentabilidad del tambo, es primordial lograr la mayor cantidad de vacas inseminadas en el menor tiempo posible. Tanto las vacas que fallan en la concepción como aquellas que padecen de pérdidas embrionarias son en las que hay que focalizar los esfuerzos para detectarlas e inseminarlas nuevamente en forma rápida y de esta manera acortar el intervalo de tiempo entre el parto y la nueva concepción. Los objetivos de este estudio fueron 1) validar la ultrasonografía (USG) Doppler para la detección de vacas no gestantes a los 19-20 d post-IA y 2) determinar las pérdidas embrionarias entre los 19 y 34 días de gestación en vacas de tambo. Al día 19-20 post IA, se evaluó la perfusión sanguínea del cuerpo lúteo (CL-FS) por USG Doppler en modo color y en modo power en todas las vacas ingresadas en el estudio (n= 131). En la misma visita también se evaluó la ecotextura uterina mediante USG en modo B para determinar si dicha evaluación podría aportar datos que mejoraran el diagnóstico de no-preñez al día 19-20 post IA. Además, se obtuvieron muestras de sangre para la medición de progesterona (P4) y para determinar la expresión de genes relacionados con la gestación temprana (genes estimulados por el interferón tau; ISG) mediante PCR en tiempo real. Finalmente se realizó el diagnóstico de gestación a los 33-34 d post-IA mediante USG en modo B. El modo Color de la USG Doppler mostró alta precisión para detectar vacuidad al pie de la vaca usando la categoría ≤1 (escala 0-4) en todos los cuadrantes del CL. La tasa de no-preñez diagnosticada USG Doppler a los 19-20 d post-IA fue de 38,9 % (51/131), mientras que el 62,6 % (82/131)Dairy producers face important challenges threatening the system's financial results, making reproductive efficiency one of the most important. To achieve the goal of a calving per cow/year and thus maintain the profitability of the dairy, it is essential to achieve a high number of inseminated cows in the shortest possible time. Cows that fail to conceive and those that suffer embryo loss are the ones that we should focus on detecting and inseminating them promptly, shortening the time interval between calving and new conception. The objectives of this study were 1) to assess the ultrasound (USG) Doppler for the detection of non-pregnant cows at 19- 20 d post-IA and 2) to determine the embryo loss between 19-34 d of pregnancy in dairy cows. In all cows included in the study (n=131), the corpus luteum (CL-FS) perfusion was assessed by the color and the power mode of the Doppler USG. In addition, the uterine echotexture was evaluated by USG mode B to predict if, including this information, the precision of the USG Doppler at 19-20 d post AI improved. At the same time, blood samples were collected to measure progesterone (P4) and to determine the expression of genes related to early pregnancy (interferon-stimulated genes; ISG) by real-time PCR. Finally, the pregnancy diagnosis was performed at 33-36 d after IA using USG mode B. Color mode showed higher precision in detecting non-pregnant cows using category ≤1 (scale 0-4) in all CL quadrants. The non-pregnancy rate at 19-20 d post-AI was 38,9% (51/131) by CL-FS Doppler USG. In contrast, the non-pregnancy rate at 33-34d post-AI was 62,6 (82/131) by B-mode USG based on the lack of embryo visualization. The presence of a thick endometrial layer and the visualization of the uterine stratum vascular at 19-20 d post-AI was related to the non-pregnancy diagnosis at 33-34 d post-AI (p1ng/ml at 19-20 d post-AI, we detected that 28,1% of the cows potentially lost the pregnancy between 19-34 d. Doppler USG allowed the detection of non-pregnant cows between 19-20 d post-AI, and the additional B-mode ultrasound evaluation of the uterine echo structure improved the detection of non-pregnant cows. The early diagnosis using these techniques is a valuable and quick tool to be implemented in resynchronization protocols to minimize open days.Fil: Lorenti, N. S.. Practica Privada; ArgentinaFil: Madoz, Laura Vanina. Universidad Nacional de La Plata. Facultad de Ciencias Veterinarias. Instituto de Teriogenología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Rearte, Ramiro. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de La Plata. Facultad de Ciencias Veterinarias. Instituto de Teriogenología; ArgentinaFil: Quintero Rodriguez, Luis Ernesto. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de La Plata. Facultad de Ciencias Veterinarias. Instituto de Teriogenología; ArgentinaFil: Migliorisi, Ana Lorena. Universidad Nacional de La Plata. Facultad de Ciencias Veterinarias. Instituto de Teriogenología; ArgentinaFil: Jaureguiberry, María. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de La Plata. Facultad de Ciencias Veterinarias. Instituto de Teriogenología; ArgentinaFil: Gabler, C.. Freie Universität Berlin; AlemaniaFil: Drillich, M.. University of Veterinary Medicine; AustriaFil: de la Sota, Rodolfo Luzbel. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de La Plata. Facultad de Ciencias Veterinarias. Instituto de Teriogenología; Argentin

Fetuin-A in elderly: effect of genotype on serum levels

Fetuin-A is a circulating inhibitor of ectopic calcification in vivo; it is decreased in patients with chronic kidney disease and correlates with glomerular filtration rate in patients with coronary artery disease. However, serum levels of Fetuin-A has not been described in elderly. Aim of the study was to evaluate Fetuin-A serum levels together with AHSG T256S genotype in a population of healthy elderly. Serum fetuin-A levels were determined by ELISA. T256S polymorphism of AHSG gene was determined by PCR-RFLP. Serum Fetuin-A was 0,38 ± 0,13 g/l in the group composed by 206 healthy centenarians. Moreover, subjects with at least one S allele had lower Fetuin-A levels (P<001). Interestingly, Fetuin-A was associated with increasing age (r=0.159; p=0.027; adjusted for sex). Fetuin-A increases with age in successful aging, suggesting a protective effect against vascular calcification exerted by Fetuin-A. Our results confirm T256S polymorphism as a genetic determinant of plasma fetuin-A levels

Fetun-A and end stage renal disease: correlations with haemodialytic single session and genotype

Several studies showed that Fetuin-a is an extracellular calcium-regulatory protein acting as a potent inhibitor of calcium-phosphate precipitation, involved also in bone metabolism because of its high affinity for hydroxyapatite. The aim of this study is to evaluate the serum Fetuin-A levels in patients with chronic renal failure before and after a single session of dialysis and to investigate the correlation between protein serum concentrations and T256S genotype. Our results show that serum Fetuin- A levels decrease 4 hours after haemodialysis treatment and a significant correlation between 256SS homozygote and serum protein levels. Ectopic calcification and particularly intravascular calcification is inhibited by Fetuin-A and this mechanism seems to be of some importance in cardiovascular morbidity and mortality in patients with End Stage Renal Disease (ESRD)