Milk ketone bodies assessment in a local italian cow breed (Modenese) vs. Holstein and characterization of its physiological, reproductive and productive performances.

Several autochthonous cattle breeds characterized by a small territorial diffusion are farmed inNorthern Italy. The technical data show that these animals have a good reproductive performance(Communod et al 2010; Communod et al 2011), disease resistance and resilience. The objective of thisstudy was to characterize some productive, reproductive and metabolic parameters (ketone bodies)in the Italian autochthonous cattle breed Modenese, comparing them with those of Holstein andtheir crossbred (F1=Modenese x Holstein; F2=Modenese x F1) breed in the same farm in order tounderstand if there is a different metabolic picture that can influence the reproductiveperformances. Milk samples have been collected at different times of lactation (20th, 40th, 90th dayin milk) and analyzed by gas chromatography-mass spectrometry to obtain the ketone bodiesconcentration. The reproductive (Open Days Period and number of Services Per Pregnancy) andproductive (percentage and kg of protein between the 40th and 90th DIM) data have beenrecovered by the consultation of the farm registers and the APA (Provincial breeder association)data. On days open, number of services per pregnancy, % of proteins in milk, and kg of proteins inmilk; a Spearman correlation analysis was applied. In all time points, the Modenese breed showed asignificant (p<0,05) lower ketone bodies concentration. The F1, F2 and Modenese showed alsobetter reproductive performances when compared to Holstein, with 80-105 days of days open inaverage. In conclusion, the better resilience against the negative energy balance and his adverseeffects of Modenese cattle could be one of the phenomena underlying their better reproductiveefficiency

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

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

Fatty acid profile, Desaturase and Atherogenic indices in milk of Holstein Friesian and Italian autochthonous cattle breeds

In the past decades, milk has been considered a mere supplier of nutrients, although its importance was considered paramount for the development and growth of newborns, a number of aspects regarding the biological functions of milk are still unknown. Several positive functional properties of milk derive from fatty acids (FA), mainly unsaturated fatty acids (UFA), either monounsaturated (MUFA) or polyunsaturated (PUFA) fatty acids. In particular, UFAs are considered functional components of food because of their positive effects on disease prevention (FAO, 2010; Connor W.E. ,2000;  Wijendran V., 2004). The objective of this study was to characterize the fatty acid profile, the desaturase index and the atherogenic index in milk of local Italian bovine breeds (Cabannina, Varzese and Valdostana) and in a cosmopolitan breed (Holstein Friesian) during the first period of lactation. A total number of 129 cows have been enrolled (Friesian n=30, Cabannina n=30, Varzese n=30, Valdostana n=39) from three dairy farms with similar management and feeding conditions. Animals were chosen in order to have three classes of lactation stage: milk collections were carried out starting from 40±10 days (group A), 70±10 days (group B), and 130±10 days (group C). Milk samples have been analyzed by gas chromatography to obtain the fatty acid profile, on the basis of these results, the Desaturase and Atherogenic Indices were calculated. A   number   of   differences   between   breeds have been  found, in   particular local  breeds showed an higher  percentages  of  UFA,  MUFA,  PUFA,  and  a  higher  UFA/SFA  ratio, as well as lower desaturase indices (related to C14, C16 and C18) and atherogenic index, when compared to Friesian cows. The results can add further information aiming to re-evaluate an almost lost local treasure in Northern Italy.

Heat treatment of bovine colostrum: II. Effects on calf serum immunoglobulin, insulin, and IGF-I concentrations, and the serum proteome.

In-depth analysis of colostrum components has identified hundreds of proteins, but data are sparse regarding their systemic uptake in the newborn calf. Moreover, heat treatment may influence these colostral components and their absorption. Our objectives were to describe the serum proteome of newborn calves before and after colostrum feeding and the possible effects of colostral heat treatment. Newborn Holstein heifer calves (n = 22) were randomized within pair and fed heat-treated (n = 11; 60°C, 60 min) or raw (n = 11) colostrum at 8.5% of birth body weight by esophageal feeder within 1 h of birth. After the single colostrum feeding, calves were not fed until after the 8-h time point, when milk was offered free-choice. Blood samples were taken immediately before feeding (0 h), as well as 4, 8, and 24 h after feeding. Whole blood packed cell volume (%), serum Brix percentage, and plasma glucose concentrations were determined for all time points. Plasma insulin and insulin-like growth factor-I concentrations were determined by radioimmunoassay for selected time points. Serum IgA and IgG were measured by radial immunodiffusion at 24 h. The serum proteome was analyzed using nano-scale reverse-phase chromatography and tandem mass spectrometry (nano LC-MS/MS) in 0- and 8-h samples. For proteomics analysis, ratios of results for 8-h to 0-h samples were analyzed with false discovery rate adjustment. For all other outcomes, repeated-measures ANOVA was performed with the fixed effects of group, time, and their interaction, and random effect of pair. Serum Brix percentage and glucose concentrations increased over time and were independent of colostrum treatment. Serum IgG and IgA concentrations at 24 h did not differ between groups. Nano LC-MS/MS identified a total of 663 unique proteins in serum, of which 261 increased in abundance, whereas 67 decreased in abundance after feeding in both groups. Among serum proteins that increased in abundance and that were previously identified in colostrum, many belonged to those involved in immune response, coagulation, the classical complement pathway, or the antimicrobial peptide class of cathelicidins. Serum proteins that decreased in abundance and that were identified in colostrum belonged to the alternative complement pathway and the membrane attack complex. Thirty-eight proteins differed in calves that were fed heat-treated colostrum compared with those fed raw colostrum. Decreased abundances in calves fed heat-treated colostrum included several enzymes involved in glycolysis or glycogenolysis, whereas the incretin gastric inhibitory polypeptide and serum insulin were increased in this group. Our findings point to important innate immune defense pathways associated with colostrum ingestion in newborn calves. Furthermore, calves fed heat-treated colostrum showed differences in serum proteins and enzymes associated with carbohydrate metabolism

Physiological response to chemical immobilization: a case study of etorphine-azaperone in free-ranging plains zebra (Equus quagga) in Kenya

Predictable immobilization of wild zebras is challenging and there is massive variation in opiate response within different species.Etorphine combined with azaperone is considered the protocol of choice, but no studies have investigated the physiological response to this procedure of immobilization in plains zebras. Eleven free-ranging plains zebras (Equus quagga) were immobilized in Kenya using a combination of etorphine 0.019 ± 0.003 mg/kg and azaperone 0.27 ± 0.05 mg/kg administered intramuscularly with a projectile dart. After recumbency, an arterial sample was performed for blood gas analysis and physiological parameters were recorded every five minutes.Descriptive scores were given to the exertion resulting from high-speed chasing and to the quality of induction, immobilization and recovery. Diprenorphine or naltrexone were used for opioid antagonism. In all zebras, the combination induced quick inductions within 3.5 ± 0.8 minutes and provided reliable recumbencies without attempts to stand for the entire duration of the immobilization.The average heart rates, respiratory rates and mean arterial blood pressure recorded were 102 ± 42 beats/minute, 18 ± 4 breaths/minute and 145 ± 28 mmHg respectively. Arterial gas analyses demonstrated mild to severe and partially compensated metabolic acidosis and hypoxia, while electrolytes were within equids range. In particular, higher exertion levels during the chasing were significantly correlated to worse immobilization scores (p=0.008) and hyperthermia occurrence (p=0.0012) and non-significantly to more severe acidosis. Recoveries from anaesthesia were smooth, on average 121 ± 38 seconds after diprenorphine/naltrexone administration.           Etorphine-azaperone combination produced physiological alterations in free-ranging plains zebra such as tachycardia, hypertension, metabolic acidosis and hypoxemia. However, these preliminary results indicate that high-speed chase might be responsible for the physiological imbalance and that this drug combination does not suppress the compensatory response. Regardless of the metabolic status, recover from immobilization was uneventful and all zebras went back to normal behavior thereafter

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

Blood baseline values in female alpine and nera di verzasca goats reared in italy.

The Italian goat autochthonous breeds are appreciated for their milk and characteristics, especially for the rusticity, frugality, fertility and longevity. For these reasons the local goat breeds play an important role in the livestock sector, and it is important to guarantee sanitary strategies control, prevention or treatment of diseases. It is well known that the hematological parameters in goats undergo changes in relation with many factors like breed, age (Piccione et al. 2014), physiological/reproductive status environmental factors and stress (Waziri M.A. et al. 2010).  Based on these differences it is necessary to establish appropriate physiological baseline values for every single breed which could be used in the realistic evaluation physiological or pathological status of the animal (Arfuso et al. 2016). The aim of this work was to evaluate the differences between a local goat breed (Verzasca) and a cosmopolite one (Alpine) from the hematological point of view, and to establish hematological reference values. A total number of 71 female goats, of Alpine (n=37), and Verzasca (n=34) were enrolled for this study, for a total of 716 blood samples. Data were processed by a mixed model-repeated measures ANCOVA in order to evaluate the effects of breed, parity, and season, while baseline values for each breed have been calculated by evaluating the 2.5-97.5th  percentile of variables distribution.The results showed that the breeds differ in a significant manner (Table 1). Verzasca goat shows significantly higher values in the erythroid parameters, whereas the Alpine goat shows higher mean values of leucocyte count and absolute neutrophil count. A further interesting result is the neutrophil lymphocyte ratio which is 0.96  in the Alpine and 0,57 in the Verzasca.The results here presented can add some knowledge to the definition of the health status of the two breeds, evidencing some environmental and physiological variation mechanisms.