Non-invasive intestinal biomarkers: a new ELISA test for Pancreatitis Associated Protein detection in pig

Feed additives are commonly used to improve pig performance and health, but they need to be tested so new biomarkers for intestinal health, non- or minimally invasive, are under investigations.The quantification of Myeloperoxidase (MPO) and Pancreatitis Associated Protein (PAP) in feces could prove useful to non-invasively monitor intestinal health (Niewold, 2015). MPO is an enzyme that permits to quantify the number of inflammatory cells present in tissues and feces (Prokopowicz et al., 2012) , while PAP is a protein mainly produced in the small intestine with anti-inflammatory and bactericidal activity (Cash et al., 2006; Mukherjee et al., 2014). Because of the lack of a commercial ELISA kit for porcine PAP detection, the main aim of this study was to develop and validate a new sandwich ELISA test for the quantification of PAP in pig fecal samples. Our study consisted of two phases: test development and test validation. During the development phase we used polyclonal antibodies previously immunized from rabbit serum with a pure peptide containing the N-terminus of pig PAP (Soler et al., 2015). The validation of the test was then performed with fecal extraction samples derived from animals with known high or low growth performance.Moreover, the temperature stability of PAP in feces and the optimal extraction method was tested. Even if only preliminary, our results seem to show a fair relationship between fecal consistency, used as health indicator, and PAP fecal concentrations. Furthermore, no relevant differences in PAP concentration after 24h of incubation at 37 °C, 4°C or room temperature were detected.To date, the present results suggest that PAP seems to be exceptionally stable in feces and is a very promising candidate as a non-invasive (fecal) biomarker for intestinal health and growth

Plasma α-tocopherol content and its relationship with milk somatic cells count in Italian commercial herds.

This work was aimed to investigate relationship between plasma vitamin E concentration and milk somatic cell count in healthy cows in commercial herds. 49 multiparous cows from two commercial dairy herds were monitored from the day of dry off until 90 DIM. BCS was assessed and blood samples were collected at dry off, day 0, 30, 60 and 90 postpartum. Plasma was analyzed for α-tocopherol content. Quantification of NEFA, BOHB, Zn and Se was performed in serum samples. Milk production and composition was obtained from routinely test-day of Italian milk producers association. Somatic Cell Score (SCS) was calculated and included in the dataset. Analysis of data was performed using MIXED repeated and CORR procedures of SAS.We did not observe a correlation between plasmatic vitamin E and somatic cell score, and this can be explained by the low level of somatic cell score (averages 1.64 and 1.26). The lowest value of vitamin E was observed at parturition (1.64 µg/ml and 1.95 µg/ml). A significant (P<0.01) negative (-20%) correlation was observed between NEFA serum content and α-tocopherol plasma concentration. Serum selenium content was positively correlated (+42%, P<0.0001) to zinc concentration. Grouping cows on the basis of their plasma α-tocopherol content higher or lower than 3 μg/mL at dry off, SCS at 30 and 60 DIM tended to be higher in lactating animals with lower content of α-tocopherol (1.12 vs. 1.72, P=0.18 at 30d; 0.92 vs. 1.72, P=0.07 at 60d). However, plasma α-tocopherol content at dry off could be usefully correlated with somatic cell count in fresh cows

Effect of live yeast (Saccharomyces cerevisiae) administration on apparent digestibility of horses

Six mares were divided into two groups of three subjects each in a crossover design on the basis of body weight and age: Control(C)fedabasaldiet,Treatment(T)fedabasaldietand2 Control (C) fed a basal diet, Treatment (T) fed a basal diet and 2 2 g/head/d of live yeast ((S. cerevisiae 4.6x1010 CFU/day).Theexperimentaldesignwasdividedintotwo ).Theexperimentaldesignwasdividedintotwo . The experimental design was divided into two periods named period 1 and period 2 respectively of 35d each, and consisting of 3 different phases. Dur- ing each period all animals were subject to an adaptation phase of 14d (phase1); during phase 2 (18d) and phase 3 live yeast was administered (T) or not (C). Phase 3 consisted in a three days individual fecal collection period all the groups, in order to determine dry matter, organic matter, crude protein, crude fat, NDF and ADF apparent digestion rates using acid insoluble ash (AIA) as internal marker (Bergero et al., 2005). Results obtained evidenced as the administration of S. cerevisiae to mature horses resulted in increased digestibility of dry matter, organic matter, NDF, and ADF

Nuclear factor E2-related factor 2’s activation in transgenic mice fed with high dosage of fish oil.

Some fatty acids, such as CLA (conjugated linoleic acid) and n-3 fatty acids modulate immune and inflammatory response in ruminants and monogastrics; their supplementation alters fatty acids profile of meat and milk, enhancing their nutritional quality. However, it is still unclear if their addition causes oxidative damage to animals. Nuclear factor E2-related factor 2 (Nrf2) plays an important role in cellular defenses against oxidative stress, indeed it produces a rapid induction of its target genes involved in antioxidant response. The aim of the project is to investigate the activation of Nrf2 in luciferase reporter mice fed different amount of n-3 PUFA in the diet (7,5% lard, 7,5% tuna oil, 20 % lard and 20%tuna oil). Forty-eight reporter mice are divided into three groups: male, intact female and ovariectomized female. Each group is split in four subgroups fed different diets. Oxidative status will be studied monitoring Nrf2’s activation with in vivo bioluminescent imaging. The inflammatory and immune response will be assessed using calprotectin and lactoferrin levels in faecal samples that are non-invasive techniques. The trial is still in progress: on the 62nd day, animals will be sacrificed after a challenge in order to measure the different effects of diets and +/- oestrogen on stress response. Finally, the post mortem analysis will be carried on extract organs. Data obtained will be analysed using statistical procedures and results will improve the knowledge about interaction between omega-3 fatty acids and animals’ oxidative status

Feed efficiency of Italian Holstein dairy heifers in the genomic era – preliminary results

Genetic selection of dairy cattle has classically been based on fertility and productivity (kilograms of milk, protein, fat). In recent years a growing interest in characters related to health and efficiency of the animals has taken hold. The selection of animals with a high feed efficiency can bring benefits in terms of health, productivity and environmental impact. A first batch of 16 Holstein heifers (mean age 12.63 ± 2.90 months) was selected and housed in a tie-stall of the Centro Zootecnico Didattico Sperimentale (CZDS) of Lodi for the whole length of the trial (35 days). Blood samples were collected and sent to the lab to perform genotyping of the animals. Heifers were fed a composed ration of sorghum silage and total mixed ration, delivered daily on individual feeders to ensure each animal continuous access to the feed. Animals had free access to the water. Feed intake was obtained weighing the supplied ration and the residual the day after. Individual body weight (BW), body condition score (BCS), hearth girth (HG) and height at the wither (W) were measured weekly. Feed Conversion Ratio (FCR) and Residual Feed Intake (RFI) were calculated using Dry Matter Intake (DMI) data. Fecal samples were collected weekly to assess any relation between feed efficiency and acidic profile of the feces. Preliminary results show an average daily gain of  941 ± 206 grams, an increase of mean BW from 374.63 ± 17.06 (day 0) to 407.56 ± 17.06 kilograms (day 34), an increase of mean BCS from 3.97 ± 0.11 (day 0) to 4.19 ± 0.11 (day 34), an increase of mean HG from 169.25 ± 2.83 (day 0) to 177.41 ± 2.83 centimeters (day 34), an increase of mean W from 127.28 ± 1.23 (day 0) to 132.69 ± 1.23 centimeters (day 34)

Can lipid supplementation modulate inflammatory state and immune response in periparturient goats? A case study on hepatic and adipose miRNA expression

Saturated or unsaturated fatty acids compounds were administered to second-parity twins-diagnosed alpine dairy goats. Experimental groups were fed either calcium stearate (ST, n.7), fish oil (FO, n.8) or a control diet without any fat supplement (C, n.8) from one wk before (30g/head/d of fatty acids) to three wks after kidding (50g/head/d of fatty acids). ST provided 26% C16:0 and 69.4% C18:0 while FO provided 10.4% EPA and 7.8% DHA. Both ST and FO diets were formulated to be isocaloric and isonitrogenous, with the same calcium content. Previous obtained results on metabolic, productive parameters and mRNA expression of genes related to lipid metabolism and inflammatory response let us to consolidate the hypothesis that in goat lipogenesis is reduced across the transition period, if compared to cow, moreover FO can postpone or reduce lipomobilization. Based on these previous results, miRNA expression was performed on the same hepatic and adipose biopsies (collected on day -7 and 7 and 21 from kidding), as a new perspective in controlling cellular pathways, implicated in adipogenesis and metabolic and endocrine functions. We examined miR-26b and 155 for the infiltration of immune cells, miR-99a, 145 and 221 for the inflammation and lipolysis, miR-143 and 378 for pro-adipogenic function. MIXED and GLM procedures of SAS software were used for statistical analysis. No diet effect, but a time effect for miR-155 and a tendency for miR-221 were found. The increase of their expression over the time after kidding let us to speculate that goats have to face a postponed and more contained inflammation due to the lipolysis, when compared to cows

Nuclear factor E2-related factor 2’s activation in transgenic mice fed with dosage of saturated or unsaturated fatty acids using in vivo bioluminescent imaging

To counteract oxidative stress cells developed several mechanisms, including the transcription factor Nuclear Factor E2-related factor 2 (Nrf2). The aim of the study was to evaluate the activation of Nrf2 in transgenic mice fed saturated or polyunsaturated fatty acids and the anti-inflammatory effect of estrogens on organism. Forty-eight ARE CRE OMO reporter mice were divided into 3 groups, consisting of 16 animals, based on presence/absence of estrogens (ovariectomized or sham female, OVX - SH; male, MA). Each group was further split in 4 subgroups of 4 animals each and fed different diets (7.5% lard, 7.5% tuna oil, 20.0 % lard and 20.0% tuna oil). Two times a week animals were anaesthetized and injected i.p. with 100µL luciferin 15 min before the imaging session. Using the Living Image Software, photon emission was mapped for selected body areas. On day 70, animals were sacrificed after a challenge with Sodium Arsenite. Specific organs were dissected and immediately subjected to ex vivo imaging session. MIXED and GLM procedures of SAS software were used for statistical analysis. Dietary treatments did not affect body weight and feed intake as well as Nrf2 expression in both pre- and post-challenge phases, with the exception of the abdominal region (P=0.031 pre-challenge); in this area, during the pre-challenge phase, OVX showed lower Nrf2 activation (P<0.001). Ex vivo results outlined a significant effect of the challenge on all the considered organs (P<0.001), while OVX subjects had higher Nrf2 expression on urinary bladder and kidney (P<0.05) and high fat diet increased Nrf2 in urinary bladder (P<0.05). The present trial shows how saturated or polyunsaturated fatty acids supplementation in the diet do not exert significant effects on oxidative stress in mice, but confirms the protective role of estrogens under physiological condition

Use of Electrical Coductivity Sensors to monitor Health Status and Quality of Milk in Dairy Goats

Intramammary infection (IMI) can adversely affect, in dairy goats, milk quality and milk yield leading to high economical losses. Although somatic cell count (SCC) and microbiological tests could be valid approaches to detect IMI, other methods of IMI early detection may be useful to detect infected animals and to improve milk quality.The aim of this study was to test a new multivariate model developed with the fuzzy logic technology and based on the milk EC - acquired on-line for each gland by dedicated sensors - and on new qualitative and quantitative indexes derived from the spectrum of the recorded signals.Results obtained showed that the fuzzy logic model tested could achive better results than those already reached in dairy goat research. Nevertheless, further experiment and more field data could be useful to reach the best possible accuracy that this multivariate approach could show

Transcriptional regulation of lipid metabolism and inflammation in transition dairy goats by fish oil and stearate

To better understand the interaction between saturated or unsaturated fatty acids and its effect onexpression of genes involved in subcutaneous adipose tissue metabolism, 23 second parity alpine dairygoats were enrolled in the experiment and fed either a non fat-supplemented basal diet (C; n=8), thebasal diet supplemented with stearic acid (ST; n=7) or the basal diet supplemented with fish oil (FO;n=8). 30g/head/d supplemental fatty acids during the dry period and 50g/head/d during lactation weredelivered starting one week before parturition up to 21 days in milk. Subcutaneous adipose tissuesamples were harvested at day -7, 7 and 21 relative to kidding and mRNA levels of genes involved ininflammation were measured via qPCR. Data were analyzed using the MIXED procedure of SAS. Nosignificant effects for treatment were observed, however eight genes were significant for time. HP andSAA3 expression peaked at day 7 postpartum, to then return at prepartum level around 21 d relative tokidding, while IL8, IL10, and IL18 expression constantly increased along the transition period. Vice versa,expression of IL1β, IL6R, and RXRA decreased in response to kidding, with a subsequent increase at day21. The obtained results led us to hypothesize that goats face a postponed lipomobilization afterkidding, probably related to their reduced production. The next step will involve the analysis of miRNArelated to immune cell infiltration, adipocyte inflammation and lipolysis and positive regulation ofadipogenesis to better understand the complex network of lipid metabolism in periparturient goats

The beneficial role of Probiotics in monogastric animal nutrition and health

The practice of probiotics in farm animals' health and production has increased significantly over the last 15 years. Probiotics are defined as live microorganisms that can confer a health benefit for the host when administered in appropriate and regular quantities. The isolation and identification of microorganisms is the first step in the selection of potential probiotics from gut, feces and milk of respective animals. The present molecular techniques mainly genomic and proteomic-knowledge based are employed to identify, characterize probiotics. The ability to examine fully sequenced genomes has accelerated the application of genetic approaches to elucidate the functional roles in the selection of new and specific probiotics. Identification of suitable probiotics may prove to be the next step to decrease the risk of intestinal diseases and reduce specific microbial disorders, as well as demonstrating their role in the production performance of animals, safety and wholesomeness of animals' meat evidencing consumer's protection. The mechanisms of action of probiotics include the inhibition of pathogen growth by competition for nutritional sources and adhesion sites, secretion of antimicrobial substances and toxin inactivation. Consequently, the primary interest in the application of probiotics has been in the prevention and treatment of gastrointestinal infections and antibiotic-associated animals' diarrheal diseases. In this review, the most important benefits of probiotics upon the gastrointestinal microbial ecosystem in monogastric animals (equines, pigs, veal calf and poultry) are described, as well as their implications in terms of animal nutrition and health. Additional knowledge on the possible mechanisms of action is also provided