Blood thyroid hormones, insulin and leptin, metabolites and enzymes in transition dairy ewes, as affected by dietary linseed and physiological stage.

This study investigated the effects of dietary linseed and physiological state on blood concentrations of some metabolic hormones and indicators, in transition dairy ewes. From 21 d before lambing to 60 d post-partum, ewes were provided with one of three isoenergetic and iso­nitrogenous pelleted concentrates, without (CTR, n = 21), or with 100 (EL-10, n = 22) or 200 g/kg (EL-20, n = 22) of extruded linseed. Animals were fed alfalfa hay ad libitum and had access to mixed pasture. Plasma thyroid hormones and insulin concentrations were not affected by diet and significantly changed by time. The last week of pregnancy T3 (2.27 ± 0.15 ng/mL) and T4 (102.63 ± 2.85 ng/mL) were lower than at 4 weeks before lambing (T3: 3.27 ± 0.27 ng/mL; T4: 125.89 ± 2.63 ng/mL). After lambing, T3 rose to be highest at 4 and 8 weeks of lactation (3.41 ± 0.22). Mean insulin peaked 2 weeks after lambing (0.31 ± 0.02 ng/mL) vs. late pregnancy (0.22 ± 0.01 ng/mL) and progressing lactation (0.18 ± 0.01 ng/mL). Plasma Leptin concentration (2.43 ± 0.03 ng/mL) was not affected by diet nor time. All the blood metabolites and enzymes investigated showed significant time x treatment interaction. Differences of several haematological parameters were found in EL vs. CTR (cholesterol, triglycerides, total protein, bilirubin, ALP, ALT), however, in most cases the values were fairly within the reference physiological ranges. Metabolic hormones are confirmed to be mainly linked to the different physiological states, energy intake and variations of energy balance, without clear effects by different sources of energy and quality of dietary lipids.

Dietary Supplementation of Olive Mill Waste Water Polyphenols in Rabbits: Evaluation of the Potential Effects on Hepatic Apoptosis, Inflammation and Metabolism through RT-qPCR Approach.

Agro-industrial processing for the production of food or non-food products generates a wide range of by-products and residues rich in bioactive compounds including polyphenols. The concentration of these by-products is sometimes higher than in the original raw material as in the case of olive mill waste water (OMWW), one of the main by-products of olive oil extraction. Polyphenols are secondary plant metabolites that regulate the expression of specific inflammatory genes, transcriptional factors and pro/anti-apoptotic molecules, thus modulating the signaling pathways essential for cell health and homeostasis. The liver plays a key role in regulating homeostasis by responding to dietary changes in order to maintain nutritional and physiological states. In this study a nutrigenomic approach was adopted, which focuses on the effects of diet-health-gene interactions and the modulation of cellular processes, in order to evaluate the expression of the genes (AGER, BAX, COX2, IL1B, PPARA, PPARG, SIRT1, TNFA) involved in these interactions in the livers of rabbits fed with a diet supplemented with OMWW (POL) or without supplements (control, CTR). The RT-qPCR analysis showed the down-regulation of SIRT1, TNFA, AGER, BAX and PPARA transcripts in the POL group compared to the CTR group. These results show that OMWW dietary supplementation prevents cell death and tissue deterioration in rabbits.

Effects of Dietary Polyphenols from Olive Mill Waste Waters on Inflammatory and Apoptotic Effectors in Rabbit Ovary.

The aim of this study was to evaluate the effect of dietary polyphenols on the expression of the effectors involved in inflammation and apoptosis in rabbit ovary. New Zealand White female rabbits were fed a basal control diet (CTR), or the same diet supplemented with a polyphenolic concentrate (POL, 282.4 mg/kg) obtained from olive mill waste waters. The follicle counts and the relative mRNA (RT-qPCR) and protein (immunohistochemistry) expression of the effectors involved in inflammation (cyclooxygenase-2; interleukin-1beta; tumor necrosis factor-alpha, TNFA) and apoptosis (BCL2-associated X protein, BAX), detected in the ovaries of both groups, were examined. The POL diet increased the primary and total follicles number. Cyclooxygenase-2 gene expression was higher (p < 0.05) in the POL group than in the CTR group, whereas BAX was lower (p < 0.05) in POL than CTR. Immunohistochemistry revealed the presence of all the proteins examined, with weaker (p < 0.05) COX2 and BAX signals in POL. No differences between the CTR and POL groups were observed for IL1B and TNFA gene and protein expression. These preliminary findings show that dietary polyphenols modulate inflammatory and apoptotic activities in rabbit ovary, regulating cyclooxygenase-2 and BAX expression, thus suggesting a functional involvement of these dietary compounds in mammalian reproduction.

Differential Effects of Dietary Oregano Essential Oil on the Inflammation Related Gene Expression in Peripheral Blood Mononuclear Cells From Outdoor and Indoor Reared Pigs.

Intensive farming systems represent a stressful environment for pigs and negatively influence neuroendocrine functions, behavior, and performance. Outdoor farming is an alternative option, which is thought to imply several beneficial effects for the animal. Dietary essential oils are known to be an innovative strategy to improve pig health and performance, and oregano essential oil (ORE) possesses beneficial effects due to its antimicrobial, anti-fungal, and antioxidant properties. We tested the effect of dietary ORE on peripheral blood mononuclear cells (PBMCs) in 36 growing pigs, either reared under indoor or outdoor conditions. Quantitative real-time PCR (RT-qPCR) assay was used to evaluate the effect of diet (control vs. ORE) and the time of sampling (T1-120 days vs. T2-190 days) on the expression of inflammatory and immune-related genes (TNF, IL1ß, IL8, IL18, IL10, IL1RN, STAT3, HSP90, ICAM-1, and NFKB1). Under outdoor condition, the majority of transcripts were upregulated (p < 0.05), assuming a general inflammatory status (TNF, HSP90, NFKB1, IL1ß, and STAT3). However, an interaction between diet and the farming system was observed: HSP90, NFKB1, and STAT3 were downregulated (p < 0.05) in the outdoor reared pigs when fed the ORE diet. Our study showed that bioactive compounds of ORE exert their activity, especially when the animals are exposed to stressful stimuli. Dietary ORE can be an acceptable strategy to help pigs tolerate the stress related to the harsh, outdoor, rearing conditions.

Dietary Supplementation with Olive Mill Wastewater in Dairy Sheep: Evaluation of Cheese Characteristics and Presence of Bioactive Molecules.

The aim of the study was to define the chemical characteristics, antioxidant capacity, oxidative status, sensory properties, and the presence of polyphenols in ovine cheese obtained after dietary administration of spray-dried olive mill wastewater (SDP). SDP is a waste from olive oil production rich in bioactive molecules obtained by further processing the olive mill wastewater through a spray-drying system. Thirty-six sheep were randomly assigned to two experimental groups that received a standard diet based on hay and concentrate. The concentrate fed to the SDP group was supplemented with SDP at a rate 25 g/kg (as fed). The trial lasted 9 weeks. Milk from the two treatment groups was separately collected and used for manufacturing cheese. Cheese quality parameters and proximate composition were not affected by the dietary treatment, whereas the antioxidant status and oxidative stability of cheese were positively affected. Polyphenol analyses in cheese were performed through liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). The concentration of hydroxytyrosol and tyrosol, and their sulphate metabolites, were higher in cheese from supplemented sheep. These findings suggest that polyphenol metabolites can play a major role in the beneficial effects observed in food produced from sheep fed SDP.

Plasmatic Profile of Pregnancy-Associated Glycoprotein (PAG) during Gestation and Postpartum in Sarda and Lacaune Sheep Determined with Two Radioimmunoassay Systems.

This study was carried out to determine ovine pregnancy-associated glycoprotein (oPAG) levels in the plasma of Sarda and Lacaune ewes throughout gestation and in the first month postpartum, using two heterologous radioimmunoassays (RIA-706 and RIA-srPool) and to study the correlations between PAG levels and fetal gender and number. On Day 18 of pregnancy, PAG concentrations were detected in 90.1% and 80.8% of Sarda pregnant ewes with RIA-706 and RIA-srPool, respectively; and in 90% and in 75% of Lacaune pregnant ewes with RIA-706 and RIA-srPool, respectively. From Day 30, PAG concentrations were detected in all pregnant ewes by using both RIA methods. In the postpartum period, the PAG concentrations in Sarda ewes decreased rapidly reaching minimal levels (<1 ng/mL) on day 28 using both RIA-706 and RIA-srPool. In Lacaune ewes, PAG-706 levels were higher than PAG-srPool from parturition until the last day of observation (Day 28 postpartum). It was also observed that mean concentrations were higher in multiple than in single pregnancies in Sarda and Lacaune ewes. Moreover, due to the rapid disappearance of PAG concentration following lambing, a cut-off limit in postpartum animals was not required as a means for detecting a new pregnancy.

Immuno- and glyco-histochemistry as a tool to evaluate the oregano supplemented feed effects in pig gut.

Among oregano properties, its antioxidant and antibacterial effects are particularly interesting. Oregano is also able to induce a higher glycoconjugate production in gut, creating a physical barrier against microorganisms. This study evaluated the effects of adding an aqueous extract of oregano (OAE) to the diet of two homogenous groups of pigs during the finisher phase. The diets were as follows: control commercial diet (CTR group) and CTR diet supplemented (2 g/kg) with OAE (O group). Samples of ileum and caecum from the two groups were examined by conventional histochemistry to analyze complex carbohydrates and by immunohistochemistry to detect Bcl-2 Associate X protein (BAX), an indicator of oxidative stress. Glyco-histochemistry showed significant differences between the two groups. Immunohistochemistry revealed a lower presence of BAX in O group. The OAE supplementation improved the production of glycoconjugates, able to enhance in pig the protection of intestinal mucosa by means of direct and indirect defense actions. The reduced BAX immunostaining observed in O group may be an indicator of enhanced antioxidant action promoted by oregano. The results of this study can be used in further research to identify ways to improve endogenous defence ability, with the aim of reducing antibiotic use and preventing antimicrobial resistance.

Oregano Feed Supplementation Affects Glycoconjugates Production in Swine Gut.

This study evaluated the effects of adding oregano aqueous extract (OAE) to the diet of pig slaughtered at finisher stage. Study was performed to identify glycoconjugates and evaluate the oxidative stress levels in the duodenum and colon intestinal tracts. Glycohistochemistry was performed by staining with Periodic acid-Schiff (PAS), Alcian blue (AB) pH 2.5, AB-PAS, AB pH 1, AB pH 0.5, low iron diamine, and high iron diamine. Serial sections were pre-treated with sialidase V before staining with AB pH 2.5 (Sial-AB) preceded or not by saponification. To study oxidative stress, an immunohistochemical analysis was applied to investigate the presence of the oxidative stress target molecule Bcl-2 Associate X protein (BAX). Findings show that oregano aqueous extract supplementation improves the production of the secretion glycoconjugates involved in direct and indirect defense, thus enhancing the protection of the pig intestinal mucosa. Moreover, the reduced BAX protein immunostaining observed in both duodenum and colon of swine of the oregano-supplemented group respect to that observed in the control group suggests an enhanced antioxidant action by oregano adding. Findings could be useful for other studies aiming to reduce antibiotic use and prevent antimicrobial resistance.

The effects of feeding supplementation on the nutritional quality of milk and cheese from sheep grazing on dry pasture.

The effect of feeding supplementation on the nutritional characteristics of milk and cheese was studied in dairy sheep grazing on low mountain dry-grasslands during summer in typical sub-Mediterranean conditions of aridity. The control group (CG) of 25 sheep grazed on grass, while the experimental group (EG) of 25 sheep grazed on grass and received 600 g a day of a barley and corn mixture. Daily milk production showed a less pronounced decrease in EG than in CG (p ˂ 0.0368). After one month of supplementation, the concentrations of retinol and α-tocopherol in milk and cheese from EG were higher than CG (p < 0.05). Supplementary feeding had a positive effect on the fatty acid composition of the sheep milk. For the first time, positive effects on the volatile composition were found in EG cheese, displaying lower percentages of carboxylic acids associated with the pungent and rancid odour with respect to CG.

Apelin system detection in the reproductive apparatus of ewes grazing on semi-natural pasture.

Apelin (APLN) is an adipokine with pleiotropic effects involved in the regulation of metabolic, cardiovascular, immune, and electrolyte balance function. Recent studies demonstrated a pivotal role in the regulation of male and female reproduction. APLN and its receptor (APLNR) were found in the hypothalamic-pituitary-gonad axis tissues, regulating gonadotropin release and steroidogenesis. However, to date, there are no studies that describe APLN system in the reproductive apparatus of the sheep. The study was performed on 10 Comisana x Appenninica adult dry ewes reared in a semi-natural pasture. Organ samples were collected from five animals in the two pasture functional phases: after maximum pasture flowering (Group 1) and after maximum pasture dryness (Group 2). Experiments were devised to characterize the gene expression and protein localization of the APLN/APLNR system in ewe reproductive apparatus; in addition, the concentration of plasma APLN was evaluated during the trial. Through immunohistochemical analysis, a positive staining for APLN was observed in the large luteal cells, in the epithelial cell coat of the ampulla, in the uterus epithelial lining and in the uterine glands. APLNR was observed in the granulosa cells, in the large luteal cells, in the secreting cells of the ampulla, in the uterus epithelial lining and uterine glands. The transcripts for APLN and APLNR were evidenced in all organ tissues examined. The highest level of APLN mRNA was detected in the Group 2 ewes in the luteal phase of the ovarian cycle compared to Group 1 ewes in the anestrous one. The relative content of APLN transcript was respectively twofold higher in the ovary (P < 0.05) and uterus (P < 0.05) and threefold higher in the ampulla (P < 0.05) in the Group 2 vs Group 1. The same trend of APLN transcript was evaluated for APLNR mRNA in uterus (P < 0.05) and ovary (P < 0.05). No difference was evidenced between Group 1 and Group 2 for APLNR mRNA levels. The plasma APLN level was fairly constant during the trial period. In conclusion, the present data suggest that the apelinergic system is involved in the reproduction function of ewes, being differentially distributed and expressed in the organs of the reproductive apparatus of ewes; these variations could be related to the sexual cycle and to the cyclic activity of the reproductive apparatus.

Immunohistochemical identification of resistin in the uterus of ewes subjected to different diets: Preliminary results.

Resistin is a polypeptide hormone of the adipokine-family, primarily, but not exclusively, produced by the adipose tissue. Recent studies suggested that resistin may affect the male and female reproductive activity. The study aim was to immunohistochemically evaluate the presence and distribution of resistin in the ovine uterus. Uterine samples were collected from two groups of ewes at the end of an experimental trial during which the animals of the first group (CTRL) were fed only by grazing while those of the second one (EXP) were supplemented with barley and corn. Using a monoclonal antibody against resistin, tested by Western Blot, the immunopositive reaction was identified in the cytoplasm of epithelial lining cells and uterine glands. The endogenous production of resistin seemed to be affected by different diet, as evidenced by staining differences between the CTRL and EXP groups. Our findings support the existence of a peripheral resistin system in the sheep uterus. It is possible that this system is involved in the functionality of the uterus, which is also affected by the animal's nutritional status.

Selenium maintains Ca2+ homeostasis in sheep lymphocytes challenged by oxidative stress.

Selenium (Se) is an essential element in human and animal diets, based upon a widespread range of beneficial effects that are primarily due to its antioxidant properties. While Se can be associated to anti-cancer and anti-diabetic activities, reproductive efficiency, and enhancement of the immune system, the mechanistic details of the corresponding biological processes are still largely elusive. To avoid deficiencies and increase bioavailability, Se it is generally supplied to livestock through Se-supplemented feeds or forage plants fertilized with inorganic Se. While the relationship between Ca2+ and ROS (reactive oxygen species) is well known, only a few studies have addressed the possible involvement of Se in the control of cytosolic Ca2+ in oxidative stress. The results on Ca2+ homeostasis were obtained adding exogenous Se in the form of SeO42- to sheep lymphomonocytes cultured in vitro. In particular, Se strongly attenuated 1mM H2O2-induced alteration of intracellular [Ca2+]C as well as the entry of extracellular Ca2+ into the cells with comparable EC50 values for sodium selenate accounting to 1.72 and 2.28 mM, respectively. In an ex vivo trial, it was observed that Ca2+ homeostasis can effectively be rescued in sheep lymphomonocytes exposed in vivo to a Se concentration of approximately 1.9 mM, that was achieved by feeding sheep with olive leaves previously sprayed with 500 mg/plant Na-selenate. Thus the results obtained suggest that the mode of action of selenium markedly influenced Ca2+-related signaling events. Furthermore, results clearly reveal that the protective effect of Se on Ca2+ homeostasis under oxidative challenge can be clearly and effectively achieved through an appropriate dietary regimen obtained also in a circular economy logic using pruning of olive trees treated to reduce tree drought stress.

Microbiological, chemical-physical and sensory characteristics of Fabriano salami from pigs fed Oregano vulgaris extract.

This study aimed at evaluating the effect of Oregano vulgaris extract supplementation of heavy pig diet on the microbiological, chemical-physical and sensory characteristics of Fabriano salami. Fabriano salamis were produced processing meat derived from pigs belonging to two dietary groups of 16 pigs each: CTRL group fed a commercial pelleted feed, and OR group fed the CTRL diet integrated with 0.2% of oregano extract. Ten salamis per group were collected at 0, 7, 20 and 45 days of ripening and analysed for total viable count, enumeration of Enterobacteriaceae, Enterococcus spp, coagulase negative and positive staphylococci, Lactobacillus spp., Lactococcus spp., presence of Salmonella spp. and Listeria monocytogens, pH, water activity (aw) and colour (CIE L*a*b* colour system). At the end of the ripening (day 45) chemical composition (AOAC, 1990), total antioxidant capacity (ORACFL), thiobarbituric reactive substances (TBARS), total phenolic content (TPC) (Folin-Ciocalteu method) and consumer tests were performed. The integration of oregano extract in pig diets did not interfere with the microbial evolution, chemical composition, pH and aw. After 20 days of ripening the OR salami were redder then CTRL samples, which were also yellower at the end of ripening. Furthermore, the diet has significantly improved the oxidative status, the polyphenolic content and antioxidant power in OR salami. The consumers gave a higher score to OR salami in informed condition.

Influence of Maternal and Postweaning Linseed Dietary Supplementation on Growth Rate, Lipid Profile, and Meat Quality Traits of Light Sarda Lambs.

The effects of dietary extruded linseed (EL) on growth performance, meat quality, and lipid profile of Semimembranosus and Longissimus lumborum muscles of 81 Sarda lambs were studied in a 3 × 3 design: EL content (0%, 10%, and 20%) of maternal dietary concentrate fed from 20 d to parturition to 60 d of lactation and EL content (0%, 10%, 20%) of lamb concentrate fed after weaning for 30 d. The basal diet was composed of alfalfa and meadow hay during pregnancy and alfalfa hay during lactation. At slaughter, carcass and meat quality were evaluated. Sensory quality of Semimembranosus from 0% and 20% EL lambs was assessed. Both maternal and postweaning diets affected growth performance, with higher body weights recorded with the 10% EL concentrate. Carcass and meat quality were not affected by diet. Saturated and monounsaturated FA decreased and n-3 polyunsaturated FA increased with increasing EL content in lamb diet. An increase in vaccenic and rumenic acid was associated with the EL content of the maternal diet. Both diets increased the n-6/n-3 FA ratio. No differences in acceptability were detected by consumers among groups. It is concluded that EL supplementation and early life nutrition can influence performance and FA metabolism in growing lambs.

Dietary supplementation of Rosmarinus officinalis L. leaves in sheep affects the abundance of rumen methanogens and other microbial populations.

BACKGROUND: Rumen microbiome has a great influence on ruminant health and productivity. Different plant extracts have been tested for their ability to modulate the rumen microbiome to improve feed digestion and fermentation. Among the evaluated plant extracts, essential oils, tannins, and saponins appeared to have positive effects on rumen protein metabolism, volatile fatty acids production, and methane and ammonia production. METHODS: The objective of this study was to evaluate the effect of rosemary (Rosmarinus officinalis L.) leaves and essential oils on rumen microbial populations. Four ruminally cannulated sheep were used in a 4×4 Latin square design fed (21 d/period): 1) a control diet composed of alfalfa hay and concentrate pellet (CTR), 2) CTR supplemented with 7 g/d/sheep of rosemary essential oil adsorbed on an inert support (EO), 3) CTR with 10 g/d/sheep of dried and ground rosemary leaves (RL), and 4) CTR with 10 g/d of dried and ground rosemary leaves pelleted into concentrate (RL pellet). Abundance of total bacteria, archaea, protozoa, and some select bacterial species or groups was quantified using qPCR, while the community of bacteria and archaea was profiled using denaturing gradient gel electrophoresis. RESULTS: No difference in abundance was noted for total bacteria, protozoa, or Ruminococcus flavefaciens between the control and the treatments, but the rosemary leaves, either in loose form or in pellet, decreased the abundance of archaea and the genus Prevotella (P < 0.001). The rosemary leaves in loose form also decreased (P < 0.001) the abundance of Ruminococcus albus and Clostridium aminophilum, while the EO increased (P < 0.001) the abundance of Fibrobacter succinogenes. The community of bacteria and archaea was not affected by any of the supplements. CONCLUSIONS: Being able to affect the abundance of several groups of rumen microbes that are known to be involved in degradation of protein and fiber and production of methane and ammonia, rosemary leaves may be used to modulate rumen microbiome and its function.

Dietary Effects of Oregano (Origanum Vulgaris L.) Plant or Sweet Chestnut (Castanea Sativa Mill.) Wood Extracts on Microbiological, Chemico-Physical Characteristics and Lipid Oxidation of Cooked ham During Storage.

The aim of this study was to evaluate the dietary effect of feeding pigs with diets enriched with sweet chestnut wood (Castanea sativa Mill.) or oregano (Origanum vulgaris L.) extract on the microbiological and chemical characteristics of cooked pork ham. Three groups of 10 pigs were fed with a control diet (CTRL), with the CTRL diet enriched with 0.2% of oregano extract (OR) and with the CTRL diet enriched with 0.2% of sweet chestnut wood extract (SCW), respectively. Six cooked hams per group were produced, sliced and packaged under a modified atmosphere (N2:CO2=80:20) and stored at refrigeration temperature (4±1°C). Three packages per cooked ham were sampled for analyses at three different storage times (0, 10 and 20 days). At day 0 time, antioxidant capacity of the products (ORACFL assay) and chemical composition were performed. At each sampling time, from all the samples the following analyses were performed: total microbial count (TMC), lactic acid bacteria count (LAB), Enterobacteriaceae count, Listeria monocytogenes, pH value, colour coordinates (L*, a*, b*), total basic volatile nitrogen (TBVN) and thiobarbituric reactive substances (TBARs) determinations. No differences in TMC, LAB and Enterobacteriaceae count, pH, TBVN, chemical composition and L* values were registered between the three groups at all the sampling times considered. No Listeria monocytogenes was detected in the samples tested. Significant differences were registered for ORACFL at 0 days, a* and b* values and TBARs value at 10 and 20 days of storage, with higher values for ORACFL, a* and b* values and lower values for TBARs in SCW and OR than CTRL. No antimicrobial effect could be recorded for OR and SCW but a higher oxidative stability, also highlighted by the colour maintenance, was observed in both OR and SCW.

Influence of manufacturing procedure on the compositional and sensory properties of n-3 fatty acid-enriched pecorino cheese.

Pecorino cheeses were produced using two different manufacturing procedures (either from raw milk or from milk previously subject to a thermisation at 65 °C for 10 s with the addition of starter culture) using the milk obtained from three groups of ewes fed three different concentrates: (1) control, (2) control enriched with 100 g/kg of extruded linseed, and (3) control enriched with 200 g/kg of extruded linseed. All concentrates were administered with alfalfa hay ad libitum. The extruded linseed-enriched diets increased the concentration of n-3 fatty acids, conjugated linoleic acid, and monounsaturated and polyunsaturated acids in raw milk and thermised milk cheese and decreased the concentration of saturated fatty acids. Changes in the manufacturing procedure did not influence the fatty acid composition of the cheeses but markedly influenced their sensory properties. In particular, heat-treating the milk and adding a starter reduced the differences in cheese odour, flavour and toughness induced by the diet when raw milk was used. Cheeses made with thermised milk and the addition of starter culture were more uniform and obtained a higher score in the preference test compared with the corresponding raw milk cheeses. An appropriate manufacturing process, therefore, can be a possible strategy to obtain cheeses with improved health-promoting properties and an unaltered acceptance level by consumers.