Automation in 3D cellular system in Live-Imaging with Microfluidic Technology CELLviewer®

Differences observed when comparing cell cultures in 2D and 3D is morphological dissimilarity and their evolution over time. Cells grown in a monolayer tend to flatten on the bottom of the plate by adhering and spreading on the horizontal plane without expanding into the vertical dimension; § Mitochondria are involved in crucial cellular tasks controlling the cell cycle and growth such as cell signaling, differentiation, and death. Damage to and subsequent dysfunction of mitochondria play a role in various diseases like diabetes, myopathy and other systemic disorders; § CELLviewer® enables the simultaneous 3D cell culture and live cell imaging as well, featuring microfluidics and time-lapse multicolour epifluorescence microscopy; § Single cell tracking in 3D space is now possible and is combined with subsequent biochemical analyses of individually tracked cells, keeping their identity traceable with CELLviewer® system; q Jurkat (ATCC) Cells grown at 37°C and 5% CO2; q Medium RPMI 1640 soil (Gibco, Life Technologies, Thermo Fisher Scientific), with 2 mM of L-glutamine, 10% FBS, 100 units/mL of penicillin and 100 mg/mL of streptomycin; q MitoGreen (PromoKine, PromoCell) incubated for 20 minutes in the dark at 37°C with MitoGreen 200 mM; q The sample is then piped inside a 50ml Falcon tube closed with a 50ml CELLviewer® DOCK and flowed inside the cartridge chamber; q CELLviewer® automatically captures sample images in Brightfield channel and GFP channel; q ImageJ software was used for image analysis using the Measure function to calculate the diameter of a single cell; q 3D surface plot plug-in to display in 3D the distribution of the intensity of spatial fluorescence; Staining of mitochondria with fluorescent dyes, antibodies or fluorescent molecules can greatly facilitate studies of their function and distribution and the viability of cells in healthy and diseased individuals. The preliminary experience conducted with CELLviewer indicates that this equipment responds to the needs of individual operators as it consists of a synthesis of different integrated tools, which works both with manual and automated control. A microfluidic system has been developed and demonstrated that the 3D model can locate the 3D model spatially, it's possible to carry out experiments in direct time in terms of physiology, toxicology and clinical pharmacology. The entire automated system allows full autonomy and protocol management thanks to the software making the operator free to conduct other work, thus increasing the productivity of his project. In summary, the proposed microfluidic technology can serve as a new platform approach, which has the potential to advance studies at the cellular level. Single-cell Jurkat cells was isolated and imaged for 4 and 7 hours respectively and intensified labelling of the mitochondria and fluidic transport were observed over time. CELLviewer® can obtain detailed images of current cellular morphology with resolution and high-quality data; employing time-lapse imaging can be achieved, the evolution of cells and their 3D morphology

Serum level of hormone and metabolites in pregnant rabbit does

The aims of this study were to compare the hormones and metabolites serum levels and the reproductive performances of nulliparous (n=100) and primiparous pregnant does submitted to artificial insemination (AI) 11 days post-partum. On the day of AI, all the does were weighed and the sexual receptivity was evaluated. The kits were weaned at 26 day. Blood samples were collect by punc- ture of the marginal ear vein from one day before AI until few days before the kindling and assayed for hormones and metabolites. The higher sexual receptivity and the fertility in nulliparous than in primiparous does confirmed the negative effect of lactation. Nulliparous does showed higher blood con- centration of leptine than primiparous, and in both the groups such level lowered during pregnancy, probably reflecting the reduction of the fat reserve. The insuline level increased during pregnancy in either groups as a consequence of the growing of the foetuses. In nulliparous does the cortisol, NEFA and T3 concentrations were higher than primiparous does. The glucose levels were similar in both the groups probably due to the homeostatic mechanisms controlling the glycemia. Hormonal and metabo- lite analyses represent a good tool for understanding the physiological mechanisms required to meet higher reproductive performance

MICROFLUIDIC LIVE-IMAGING WITH CELLVIEWER TECHNOLOGY TO PERFORM BIOTECHNOLOGICAL TASKS

Cells grown in a monolayer tend to flatten in the lower part of the plate adhering to and spreading in the horizontal plane without expanding in the vertical dimension. The result is that cells grown in 2D have a forced apex-basal polarity. Microfluidic Live-Imaging with CellViewer technology is an ideal solution to observe the maintenance of a cell in excellent health, trying to bridge the gap between the 2D and 3D model. In this work we propose to test the system on a single isolated Jurkat cell in the microfluidic cartridge and record the timelapse for 4 hours. After adaptive autofocus, when sliding inside the cartridge chamber, the single cell is tracked under the action of the optics and the 3D rotation was experimentally successfully achieved. Then a single cell viability assessment was used using MitoGreen-dye a fluorescence marker selectively permeable to live cells. ImageJ software was used to: calculate the diameter of a single cell, create fluorescence intensity graphs along a straight line passing through the cell, visualize spatial fluorescence intensity distribution in 3D

Energy homeostasis in rabbit does during pregnancy and pseudopregnancy

This study was conducted to evaluate the changing concentrations of metabolic hormones and metabolites in pregnant (P) and pseudopregnant (PP) rabbit does. Twenty-five New Zealand White rabbit does were submitted to artificial insemination (AI) and then classified as P (n = 15) or PP (n = 10). Blood samples were collected weekly until day 32 post AI. During pregnancy, leptin concentrations were greater on Days 14 and 21 (P < 0.05), while insulin was greater on days 21 and 32 post AI (P < 0.05) compared to PP does. The triiodothyronine/thyroxine (T3/T4) ratio was greater in the first and last week (P < 0.001); whereas, cortisol concentrations were greater in the last week of pregnancy and after parturition (P < 0.01) compared with that of PP does. Non-esterified fatty acids (NEFA) concentrations increased from day 7 until day 32 post AI (P < 0.05). Glucose concentrations were unchanged throughout pregnancy although concentrations were positively associated with litter size. These results indicate concentrations of hormones and metabolites change during pregnancy to ensure energy requirements are met for both the foetuses and the maternal tissues. Physiological hyperleptinemia, hyperinsulinemia, and changes in cortisol as well as thyroid hormones indicate there is an adaptation of metabolic functions induced by pregnancy. These adaptations could be mediated by gonadal steroids because changes mainly occur in the second half of pregnancy when the profile of the sex hormones differs between P and PP does

Preliminary Evidence of Endotoxin Tolerance in Dairy Cows during the Transition Period

The blastogenic response of bovine peripheral blood mononuclear cells (PBMCs) to lipopolysaccharides (LPS) has been investigated for a long time in our laboratories. In particular, a possible correlation between the blastogenic response to LPS and the disease resistance of dairy cows has been suggested in previous studies. Isolated PBMCs from eight cows at three different time points during the transition period (T0 = 15 days before calving; T1 = 7 days post-calving; T2 = 21 days post-calving) were cultured in the presence or absence of LPS, and the blastogenic response was assayed 72 h after in vitro stimulation. Moreover, the gene expression of proinflammatory cytokines and kynurenine pathway molecules was investigated by real-time RT-PCR on both unstimulated and stimulated PBMCs. The cows were retrospectively divided into healthy and diseased, based on the development of peripartum diseases (subclinical ketosis and placenta retention). The comparison between healthy and diseased cows suggested that healthy animals seemed to better control the response to LPS. On the contrary, diseased animals showed a much higher inflammatory response to LPS. Moreover, cows were retrospectively classified as high and low responders based on the in vitro proliferative response of PBMCs to LPS, using the median value as a threshold. Unstimulated PBMCs of low responders showed higher expression of the proinflammatory cytokines Interleukin 1-beta (IL-1 beta), Interleukin 6 (IL-6) and Tumor Necrosis Factor-alpha (TNF-alpha), compared to high responders. Our preliminary data suggest that, during the peripartum period, high responders seem to be more tolerant to endotoxins and develop a lower inflammatory response to different stressors. Instead, low responders could be more prone to the development of unwanted inflammatory conditions in response to mild/moderate stressors

Approaches to Identify Pregnancy Failure in Buffalo Cows

Simple SummaryEmbryonic mortality and pregnancy failures still represent a major issue in domestic livestock production, particularly in dairy cattle. Despite the presence of extensive work in this research area, there is still no effective, accurate and practical method able to determine timing and viability of embryo specifically during early gestation. Indeed, technologies and techniques for predicting pregnancy success must continue to be developed. The aim of this work was to find the best strategy to diagnose pregnancy failures in buffalo cows in order to improve farm reproductive management. Among the methods compared in this study (ultrasonography, progesterone, PAGs), pregnancy-associated glycoproteins (PAGs) seem to be the best marker for predicting embryonic mortality between 25 and 40 days of gestation to be utilized as a diagnostic tool to improve reproductive management in buffalo farms.The aim of this work was to find the best strategy to diagnose pregnancy failures in buffalo. A total of 109 animals belonging to a buffalo herd subjected to a synchronization and artificial insemination (AI) program were enrolled in this study. Blood samples were collected at days 0, 14, 25, 28 and 40 after AI for the determination of progesterone (P4) and pregnancy-associated glycoproteins (PAGs) by the radioimmunoassay (RIA) method. Transrectal ultrasonography was performed on day 25, 28 and 40 after AI to monitor pregnancy. The animals included in the data analysis were assigned ex post in pregnant (n = 50) and mortality (n = 12) groups. By ultrasonography, the predictive sign of mortality was the heartbeat. At day 25, the PAGs concentration was significant in predicting embryonic mortality with respect to ultrasonography and P4, at the cut-off of 1.1 ng/mL. At day 28, either PAGs, at a cut-off of 2.2 ng/mL, or ultrasonography, with no detection of heartbeat, were highly predictive of embryonic mortality. PAGs were the best marker (p < 0.05) for predicting embryonic mortality between 25 and 40 days of gestation in buffalo. Its utilization as a diagnostic tool can influence management decisions in order to improve farm reproductive management

Licium Barbarum cultivated in Italy: Chemical characterization and nutritional evaluation

Goji berries are the most cultivated fruit crop in Asian countries as they contain many nutrients and health-promoting bioactive compounds. These health-promoting properties have recently stimulated the interest of food and nutraceutical industries in Europe, so this crop has spread within Italy, which has become the largest European producer. Several works on the chemical composition and biological activities of Chinese goji berries are available. In this review, the chemical and the nutritional profile of goji berries from Licium barbarum spp. cultivated in Italy are reported

Effect of Goji Berry (Lycium barbarum) Supplementation on Reproductive Performance of Rabbit Does

Simple Summary Infectious diseases represent serious problems for the reproductive performance of livestock animals because they negatively affect not only the welfare of the animals, but also the profitability of the farm. Moreover, the European Community continues to promote the reduction of the use of antibiotics and hormones in animal breeding. In this context, it is necessary to find new nutritional approaches to reduce the negative energy balance, and at the same time, to reinforce the immune system of the animals. In this research, the effect of goji berry supplementation on the reproductive activity and productive performance of rabbits is evaluated. Lycium barbarum fruit is considered a nutraceutical natural product containing various biologically active substances that show health benefits for both humans and animals. In particular, the berry can modulate hormones and metabolites involved in energy balance and reproduction, stimulate and balance the immune system activity, contributing to the defense of the organism against pathogens. Our results suggest that the integration with goji berry in the rabbit diet at 1% affects the reproductive activity, influencing the pattern secretion of luteinizing hormone (LH) and estrogens, as well as the sexual receptivity. Moreover, the fruit induced a higher milk production, improving the productive performance of young rabbits. Goji berry shows a wide range of beneficial properties in human health, but only a few studies evaluated its effects in livestock animals. The objective of this research was to assess the effects of goji berry supplementation on the hormonal profile, productive, and reproductive performance of does. Two months before artificial insemination, 105 nulliparous does were randomly divided into three groups (n = 35) based on the dietary treatment: commercial diet (C), or a diet supplemented with either 1% (G1), or 3% (G3) of goji berry, respectively. The results showed that receptivity was higher in G1 than in the C group (p < 0.05). Trends toward significance for differences between the G1 and G3 groups in marginal means of LH concentrations (p = 0.059), and between G1 and C in LH AUC values (p = 0.078), were evidenced. Estrogen concentrations showed a more fluctuating trend but a significant interaction effect (p < 0.001). The G1 group showed higher litter weight than C at birth (p = 0.008) and weaning (p < 0.001), as well as higher litter size at weaning (p = 0.020). The G1 group also exhibited the highest mean milk production (p < 0.01). In conclusion, goji berry influenced reproductive and productive performance, probably via modulating hormonal patterns and milk production in rabbits. However, further studies are needed to validate these preliminary results

Ante-mortem and Post-mortem Inspection and Relationship between Findings in a North Albanian Pig Slaughterhouse

Simple Summary In European Union abattoirs, the safety of meat is dependent on the favorable opinion from an official veterinarian, in accordance with the current legislation. From this perspective, the feedback generated from the ante-mortem visit and the post-mortem inspection can be investigated to control the health and welfare conditions of the animals in the pre-slaughter phases. From this perspective, we evaluated the ante-mortem and post-mortem inspection outcomes of slaughtered pigs in northern Albania and correlated the results in order to gain insight into the conditions and injuries of pigs slaughtered outside the European context and to extend knowledge on the possible relationship between ante-mortem and post-mortem relief. Dyspnea and tail, skin, and ear lesions were the most frequently observed conditions before slaughter, while pleuritis, pneumonia, liver alterations, white spots on the liver, and pericarditis were the most frequent lesions after slaughter. A significant increase in the total number of post-mortem findings was also observed as the number of ante-mortem findings increased. Overall, the prevalence of the findings observed in this study falls within the broad range of the data in the literature, but additional information should be collected during meat inspection so as to better understand the relationship between ante- and post-mortem outcomes. In June 2014, Albania was granted EU candidate status, thus starting a process of compliance with the membership criteria. In this context, a modern meat inspection approach in line with the European legislation was applied to a pig slaughterhouse in northern Albania in order to investigate the ante-mortem (AM) and post-mortem (PM) conditions and the relationship between these findings. For this purpose, 3930 pigs divided into 35 batches were evaluated over a 3-month period. The most frequent AM conditions recorded were tail lesions and dyspnea (9.1%), followed by skin (8.9%) and ear lesions (8.5%), while in the PM inspections, pleuritis was the most frequently observed condition (10.2%), followed by pneumonia (8.5%), liver alterations (5.7%), milk spot liver (3.8%), and pericarditis (3.3%). With the exception of liver alterations, the other PM lesions mentioned were positively associated with lesions on the ears (OR = 1.036; p < 0.001) and skin (OR = 1.026; p = 0.011) and dyspnea (OR = 1.021; p = 0.005), confirming the link between these variables and the health and welfare conditions of pigs on farms. Overall, the evidence that emerged from this Albanian slaughterhouse can be considered in line with other European contexts, especially in light of the considerable variability in the data present in the literature