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

Effects of Diets Enriched in Linseed and Fish Oil on the Expression Pattern of Toll-Like Receptors 4 and Proinflammatory Cytokines on Gonadal Axis and Reproductive Organs in Rabbit Buck

Infections of the genital tract can perturb the fertility in humans and animals. Pathogen recognition and activation of innate immunity onset through the pattern recognition receptor activation, such as Toll-like receptor 4 (TLR4), leading to the production of proinflammatory cytokines and mediators. TLR4 is expressed both on leukocytes and nonimmune cells. Rabbit TLR4 shows great similarity to its human counterpart. Moreover, the TLR4 signalling pathway could be modulated by long-chain polyunsaturated fatty acids (LC-PUFA). The objectives of this study were (i) to determine the expression levels of TLR4 and proinflammatory cytokines in the reproductive hypothalamic-gonadal axis of the male rabbit and (ii) to evaluate if the n-3 PUFA-enriched diets can modify their expression levels in the tissues and LC-PUFA profiles in seminal plasma. Fifteen rabbit bucks (n=5/experimental group) were fed with different diets: Commercial standard (group C), rich in extruded linseed (10%, group L), and in fish oil (3%, group FO) for 110 days. TLR4, TNF-, and IL-1β mRNA were ubiquitously expressed throughout the hypothalamic-gonadal axis. However, TLR4 mRNA expression was lower in the hypothalamus than the epididymis (P<0.01), seminal vesicles (P<0.01), and pituitary gland (P<0.05). Dietary enrichment in PUFA did not modify the gene expression profile nor the histological characteristics of the tissues. Conversely in seminal plasma, rabbits fed with L and FO had lower n-6 (P<0.05), LC-PUFA n-6 (P<0.05), and n-6/n-3 ratio (P<0.05) but higher n-3 (P<0.001) and LC-PUFA n-3 (P<0.01) compared to the control group. Our study builds a map of the gene expression of TRL4 and proinflammatory cytokines in the reproductive hypothalamic-gonadal axis of the male rabbit, fundamental step for understanding the immune defence mechanisms. Diets enriched in LC-PUFA did not affect basal gene expression but modulated sperm fatty acid composition. Finally, rabbit may be an excellent animal model to study the relationship between inflammation and infertility, and the nutritional modulation of immune functions

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

Polymorphism of the MTNR1A Melatonin Receptor Gene in Goat Breeds of Northern Italy

Melatonin receptor MT1, encoded by the MTNR1A gene, is the main one involved in the seasonal regulation of reproductive activity. A correlation between this gene polymorphism and reproductive performance has been demonstrated in sheep. To date, no data about MTNR1A gene polymorphism are available regarding Italian goat breeds other than the Sarda goat. This study aimed to detect any PCR-RFLP polymorphic sites of MTNR1A using MnlI and RsaI enzymes in Northern Italian goat breeds, which are characterized by a pronounced reproductive seasonality. One-hundred-eight adult female goats belonging to four different breeds were included in the study (i.e., Frisa Valtellinese, n = 29; Orobica, n = 23; Lariana, n = 29; Camosciata delle Alpi, n = 27). Blood was sampled from each goat. Genomic DNA was extracted from each sample and the main part of exon II of MTNR1A gene was amplified by PCR and digested with MnlI and RsaI enzymes. Unexpectedly, none of the fragments were found to be polymorphic. The absence of polymorphism may be linked to the macro group of goat breeds that evolved during human migrations. Breeds of the Alpine-European strain would appear to show no polymorphism, as confirmed by our study, whereas breeds belonging to the Mediterranean-African or Asian-Middle Eastern strains do

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

Interferon-Tau in Maternal Peripheral Blood and Its Relationship with Progesterone and Pregnancy-Associated Glycoproteins in the Early Phases of Gestation in Water Buffalo

The aim of this study was to investigate the interferon tau (IFNt) concentration in the peripheral maternal blood during the early phase of pregnancy in buffalo cows and improve the knowledge on the physiological importance of circulating IFNt, evaluating the possible interaction with pregnancy-associated glycoproteins (PAGs) and progesterone (P4). Blood samples were taken from buffalo cows on day 0 (day of AI), 7, 14, 18, 28, and 40 post insemination for the IFNt, PAG, and P4 analysis and to determine the IFNt mRNA expression. The animals were categorized ex post into Pregnant, Non-pregnant and Embryo mortality groups. The interferon value was influenced by group (p = 0.003), being always higher in pregnant buffalo cows than in non-pregnant ones, while the embryo mortality group showed intermediate values between those for pregnant and non-pregnant animals. The mRNA expression of IFNt was not influenced by groups or any time points. The regression analysis that included IFNt as the independent variable showed that PAGs, from day 18 (p < 0.01), and P4, from day 28 (p < 0.05), were positively associated with IFNt values. The close associations among IFNt, PAGs and P4 demonstrate that all three molecules work together for fetal–placental well-being and pregnancy support. Unfortunately, the great individual variability in circulating IFNt makes this analysis unsuitable for early pregnancy diagnosis

Colostrum Quality in Different Goat Breeds Reared in Northern Italy

This study aimed to characterize the colostrum quality in three different local goat breeds of Northern Italy (i.e., Frisa Valtellinese, Orobica, and Lariana) and a cosmopolitan one (i.e., Camosciata delle Alpi) (n = 30 per breed), reared under traditional semi-extensive and intensive systems, respectively. Lariana showed the highest percentage of fat (10.18 ± 3.14%) and total solids (30.73 ± 4.89%) but the lowest percentage of lactose (1.87 ± 0.82%; p < 0.05); Orobica had the lowest percentage of fat (7.13 ± 2.48%), total solids (24.11 ± 5.48%), and protein (10.77 ± 4.53%) but the highest percentage of lactose (3.16 ± 0.73%; p < 0.05). This suggests that breeds which have a more pronounced meat aptitude (i.e., Frisa and Lariana) have a higher concentration of components than breeds with more dairy aptitude (i.e., Orobica and Camosciata). Uni- and multivariate analyses showed that IgG is the parameter that best differentiates local breeds from cosmopolitan ones (p < 0.01). Colostrum from Frisa goats showed the highest IgG concentration (100.90 ± 8.11 mg/mL), while the lowest concentration was in the Camosciata breed (74.75 ± 20.16 mg/mL). Finally, the highest lactoferrin concentration was in Frisa (1781.3 ± 892.6 μg/mL) and the lowest in Camosciata and Lariana (763.1 ± 357.9 and 1148.0 ± 858.6 μg/mL, respectively; p < 0.05). Differences between Camosciata and local breeds could be due to the different farming systems, in addition to the genetic characteristics. The higher quality of colostrum produced by some local goats could be an adaptive characteristic that helps the growth and survival of the kids