Hypothalamic-pituitary-adrenal axis activation and immune regulation in heat-stressed sheep after supplementation with polyunsaturated fatty acids

The aim of this study was to assess the effects of supplementation with polyunsaturated fatty acids from different sources on immune regulation and hypothalamic-pituitary-adrenal (HPA) axis activation in heat-stressed sheep. The experiment was carried out during the summer 2012. Thirty-two Comisana ewes were divided into 4 groups (8 sheep/group): (1) supplemented with whole flaxseed (FS); (2) supplemented with Ascophyllum nodosum (AG); (3) supplemented with a combination of flaxseed and A. nodosum (FS+AG); and (4) control (C; no supplementation). On d 22 of the experiment, cortisol concentrations in sheep blood were measured after an injection of ACTH. Cellular immune response was evaluated by intradermic injection of phytohemagglutinin (PHA) at 0, 15, and 30 d of the trial. Humoral response to ovalbumin (OVA) was measured at 0, 15, and 30 d. At 0, 15, and 30 d of the experiment, blood samples were collected from each ewe to determine production of T-helper (Th)1 cytokines (IL-12 and IFN-γ), and Th2 cytokines (IL-10, IL-4, IL-13), and concentrations of heat shock proteins (HSP) 70 and 90. Ewes supplemented with flaxseed alone had greater cortisol concentrations and a longer-lasting cell-mediated immune response compared with ewes in the control and other groups. Anti-OVA IgG concentrations increased in all groups throughout the trial, even though ewes in the FS+AG group had the lowest anti-OVA IgG concentrations at 15 d. The level of IL-10 increased in all groups throughout the experiment; the FS+AG group had the lowest IL-13 concentration at 15 and 30 d. The concentration of HSP 70 increased in AG ewes at the end of the experiment and decreased in FS ewes, whereas that of HSP 90 increased in FS ewes compared with FS+AG ewes. Flaxseed supplementation was found to influence in vivo HPA activation in heat-stressed sheep, resulting in increased cortisol concentrations, probably to meet increased energy demand for thermoregulation. Flaxseed supplementation also supported Th1 response via a complex cross-talk between IL-10, IL-12, and IFN-γ production.     

Peripheral blood mononuclear cell proliferation and cytokine production in sheep as affected by cortisol level and duration of stress

A large number of studies recognize glucocorticoids (Gc) as suppressors of inflammation; Gc exert an important role in coordinating the magnitude and duration of host immune responses. In the present in vitro investigation, we tested incremental levels of cortisol to verify the immunosuppressive or immunopermissive role of cortisol in sheep peripheral blood mononuclear cells (PBMC) after acute and chronic stress. Phytohemagglutinin (PHA)-stimulated PBMC were cultured for 24 h and 96 h at 37°C with 5% of CO₂ and varying cortisol levels: 10 ng/mL (baseline), 100 ng/mL (physiological poststressor), and 1,000 ng/mL [hyperactivated hypothalamic-pituitary-adrenal (HPA) axis]. The cell-free supernatants were collected for determination of IL-6, IL-1β, and IL-10 by ELISA, and the bromodeoxyuridine assay was performed on cells. Physiological cortisol concentration negatively affected the levels of IL-6 secreted by PBMC, resulting in increased cell proliferation after acute stress (24 h of incubation). However, physiological cortisol concentration exhibited a reduction in cell proliferation induced by increased levels of IL-6 secreted by PBMC during chronic stress (96 h of incubation). The cortisol concentration representing a hyperactivated HPA axis led to a reduction in cell proliferation after acute stress, which was probably induced by the elevated IL-10 production. Our results demonstrate that in sheep the effect of Gc on the immune system was related to the magnitude and the duration of stress. In particular, cortisol levels higher than physiological concentrations suppressed cell proliferation soon after acute stress. Instead, the physiological poststressor concentration of cortisol affected the immune responses in a bidirectional manner depending on the duration of the stressor.     

Milk nutrition and childhood epilepsy: An ex vivo study on cytokines and oxidative stress in response to milk protein fractions

We present a pilot study on the effects of milk protein fractions [α_S1-casein (CN), α_S2-CN, κ-CN, β-CN, and a mix of α-lactalbumin (α-LA) and β-lactoglobulin (β-LG)] from different animal species (bovine, ovine, and caprine) on pro- and anti-inflammatory cytokines and oxidative status in cultured peripheral blood mononuclear cells from children with generalized epilepsy. Peripheral blood mononuclear cells (PBMC) were obtained by density gradient from blood of 10 children with generalized epilepsy (5 males; mean age 33.6 ± 5.4 mo) and 10 controls (5 males; mean age 35.6 ± 6.8 mo). Children with epilepsy were grouped according to cytokine levels as follows: children with epilepsy having low levels of cytokines not different from those of control children (LL-EC); children with epilepsy having cytokine levels at least 5-fold higher (medium levels) than those of control children (ML-EC); and children with epilepsy having cytokine levels at least 10-fold higher (high levels) than those of control children (HL-EC). The production of tumor necrosis factor-α (TNF-α), IL-10, IL-6, and IL-1β was studied in cultured PBMC incubated with α_S1-CN, α_S2-CN, κ-CN, β-CN, and a mix of α-LA and β-LG from bovine, caprine, and ovine milks. The levels of reactive oxygen and nitrogen species (ROS/RNS) and catalase activity were assessed in cultured supernatant. In the HL-EC group, β-CN from small ruminant species (ovine and caprine) induced the highest levels of TNF-α, whereas PBMC incubated with α_S2-CN from ovine milk and the mix of β-LG and α-LA from all tested milk species had the lowest levels of TNF-α. Within the HL-EC group, production of IL-1β was higher for bovine and ovine α_S2-CN fractions and lower for caprine and ovine β-CN and κ-CN. In the HL-EC group, IL-6 was higher in cultured PBMC incubated with α_S2-CN from bovine and ovine milk than from caprine milk. The cytokine IL-10 did not differ among milking species. The highest levels of ROS/RNS were found after incubation of PBMC with the β-CN fraction in bovine milk. Catalase activity was higher in PBMC cultured with β-CN isolated from bovine and caprine milk and with α_S1-CN from ovine milk.     

Differential leucocyte count for ewe milk with low and high somatic cell count

This study was undertaken to compare flow cytometry (FC) and direct microscopic leucocyte count (MDLC) for the differentiation of macrophages, lymphocytes and polymorphonuclear leucocyte (PMN) and to evaluate leucocyte distribution in ewe milk with low and high somatic cell count (SCC). Milk samples were grouped for somatic cell count in low SCC (LSCC) when the content was lower than 5·00 × 10(5)/ml and high SCC (HSCC) when the content was higher than 1·00 × 10(6)/ml. No differences were found between the two methods tested suggesting that FC could be used as a routine test for rapid discrimination of leucocytes. Percentages of lymphocytes in ewe milk were higher in LSCC (50%) than in HSCC (39%) and count ranged from 273·91 ± 56·62 × 10(3) cells/ml (LSCC) to 308·90 ± 46·15 × 10(3) cells/ml (HSCC). PMN number was lower in LSCC than in HSCC (248·83 ± 46·87 × 10(3) cells/ml v. 444·38 ± 58·62 × 10(3) cells/ml); accordingly the percentage was lower in LSCC (40%) than in HSCC (57%). No differences were found for macrophages which were 36·36 ± 5·51 × 10(3) cells/ml and 39·32 ± 6·83 × 10(3) cells/ml in LSCC and HSCC, respectively. Lymphocytes in ewe milk did not vary with increased number of somatic cells and were the predominant cell type in LSCC. PMN represented the main population detected in HSCC and the correlation with SCC evidenced that this leucocyte class could be useful in differentiating ewe milk cell count, being strictly responsible for the SCC increase.     

Influence of space allowance and housing conditions on the welfare, immune response and production performance of dairy ewes

The experiment used 45 Comisana ewes, divided into 3 groups of 15. The aim was to determine the effects of two different stocking densities and two different housing conditions on welfare, and on production performance of dairy ewes. The stocking densities tested were: high stocking density (1 x 5 m2/ewe, HD group) and low stocking density (3 m2/ewe, LD group); the two housing conditions tested were: ewes housed indoors (LD group, 3 m2/ewe) and ewes allowed to use an outdoor area (LDP group, 3 m2/ewe divided into 1 x 5 m2/ewe indoors and 1 x 5 m2/ewe outdoors). At the beginning of the experiment, and then every 2 months, the cell-mediated immune status of sheep was evaluated. One month after the beginning of the experiment, and 20 d later, the ewes were injected with chicken egg albumin (OVA) to assess their humoural immune responses. Starting from the beginning of the experiment and then monthly, behavioural activities of ewes were monitored using 15-min scans. After lamb weaning, milk yield from individual ewes was measured and milk composition analysed weekly. Housing conditions (low density reared ewes indoors v. low density reared ewes with free access to an outdoor area) affected cell-mediated response, which was higher in LDP than in LD ewes. Concentrations of anti-OVA IgG were mainly influenced by space allowance, with higher antibody titres in LD than in HD ewes throughout the experiment. Both housing conditions and space allowance affected sheep behavioural activities: a greater proportion of LDP ewes displayed standing and drinking behaviours than LD ewes, and a greater proportion of LD ewes was observed walking than HD ewes. Ewes allowed access to the outdoor area had a higher protein content and lower somatic cell count in their milk, whereas reduced space allowance led to a reduction in milk yield and an increase in somatic cell count of milk. Results indicate that both increased space allowance and availability of outdoor area can improve the welfare and production performance of the lactating ewe.     

Effect of information about animal welfare, expressed in terms of rearing conditions, on lamb acceptability

The present study was aimed to assess the effect of information about the farming system and welfare of artificially (AR) and ewe-reared lambs (ER) on meat acceptability. An 84-consumer panel was asked to give three types of evaluation for both products: tasting without information (perceived liking); information without tasting (expected liking); tasting with information (actual liking). For both products, the expected liking was significantly different from the perceived liking (P<0.001), thus indicating that a disconfirmation occurred. The consumers found ER meat worse than expected (negative disconfirmation) and AR meat better than expected (positive disconfirmation). No significant difference between perceived and actual liking was observed for ER lamb (P>0.05), whereas, for the meat produced by AR lambs actual liking was lower than perceived liking (P<0.001). However, consumers did not completely assimilate their liking in the direction of expectations, as indicated by the fact that expectancy for AR lamb was significantly lower than actual acceptability (P<0.001). Mean scores of perceived liking were higher for the meat produced by AR animals as compared to ER lambs (P<0.05). Conversely, expected and actual liking scores were higher for meat from ER lambs than for meat from AR animals (P<0.001 and P<0.05, respectively). These results show that information about animal welfare, expressed in terms of rearing conditions, can be a major determinant of animal-based food acceptability, thus providing a potential tool for meat differentiation.     

Flaxseed supplementation improves fatty acid profile of cow milk

The objective of the study was to determine the effects of adding flaxseed or fish oil to the diet on the milk fatty acid profile of cows. The experiment was conducted in the summer of 2006 and involved 24 Friesian cows that were divided into 3 groups of 8 animals according to different type of fat supplementation: a traditional diet with no fat supplementation, a diet supplemented with whole flaxseed, and a diet supplemented with fish oil. Results suggested that whole flaxseed supplementation positively affects the milk fatty acid profile during summer. In particular, milk from cows receiving flaxseed supplementation showed a decrease in saturated fatty acid, an increase in monounsaturated fatty acid, and, together with the milk from fish oil-supplemented cows, an increase in polyunsaturated fatty acid content compared with milk from control cows. As expected, both fish oil and flaxseed supplementation increased the content of n-3 polyunsaturated fatty acids in milk fat. The increased dietary intake of C18:3 in flaxseed-supplemented cows resulted in increased levels of milk C18:1 trans-11 and increased conjugated linoleic acid C18:2 cis-9,trans-11 by Δ⁹-desaturase activity. Milk from flaxseed-supplemented cows together with the high conjugated linoleic acid content was characterized by low atherogenic and thrombogenic indices, suggesting that its use has less detrimental effects concerning the atherosclerosis and coronary thrombosis risk associated with the consumption of milk and dairy products. In conclusion, flaxseed supplementation improves composition and nutritional properties of milk from cows milked during times of high ambient temperature.     

Ventilation effects on air quality and on the yield and quality of ewe milk in winter

The effects of ventilation on air quality, and on the welfare and production performance of dairy ewes were assessed in a 6-wk trial conducted during the winter of 2002. Thirty-six midlactation Comisana ewes were divided into three groups of 12, which were randomly balanced for parity, time of lambing, and number of lambs suckled. Treatments were low (LOV), moderate (MOV), and programmed ventilation regimen (PROV). In LOV and MOV rooms, fans provided 10 ventilation cycles of 40 min each at a fan speed of 1 and 2 m/s, respectively. In the PROV room, the fan was programmed to maintain a 70% relative humidity. Mean ventilation rates were 23, 47, and 73 m3/h per ewe in LOV, MOV, and PROV rooms, respectively. Air concentrations of microorganisms and dust, and of gaseous pollutants were measured twice weekly. Cell-mediated immune response to phytohemagglutinin at d 1, 21 and 42, and humoral response to chicken egg albumin at d 11, 21, 30, and 40 were determined. At d 39, ewes were injected with 2 IU of porcine adreno-corticotropic-hormone/kg body weight(0.75), and subjected to blood sampling for evaluation of cortisol concentrations immediately before and 1, 2, and 4 h after adreno-corticotrophic-hormone injection. Milk yield was recorded daily. Individual milk samples were analyzed weekly for composition, renneting parameters, and somatic cell count. The LOV treatment resulted in higher air concentrations of NH3 and CO2 than the MOV and PROV treatments. Greater amounts of total and respirable dust were found in the PROV room than in the LOV and the MOV rooms. The LOV ewes had lower milk yield than the PROV ewes, lower milk casein content, and higher rate of clot formation than the MOV and PROV ewes. The ventilation regimen did not affect the immune and endocrine responses of the ewes. Results suggest that an intermittent ventilation regimen, providing a mean ventilation rate of 47 m3/h per ewe at a fan speed of 2 m/s, is required to sustain the yield and cheese-making ability of ewe milk during the winter season.     

Effect of different ventilation regimens on ewes' milk and Canestrato Pugliese cheese quality in summer

The influence of three different ventilation regimens on air pollution in sheep houses and on the quality of ewe milk and of Canestrato Pugliese cheese was investigated during the summer season. The experimental treatments were low ventilation regimen (VR=35 m3/h per ewe) split in 30-min ventilation cycles (LOV-30); moderate ventilation regimen (VR=70 m3/h per ewe) split in 30-min ventilation cycles (MOV-30); moderate ventilation regimen (VR=70 m3/h per ewe) split in 60-min ventilation cycles (MOV-60). The LOV-30 milk had higher microbial load and bulk milk somatic cell count (BMSCC) and resulted in a weaker casein matrix in the curd compared with the MOV-30 and MOV-60 treatments. At 45 d of ripening, the LOV-30 cheeses had a lower casein content and higher non-casein nitrogen (NCN) and water-soluble nitrogen (WSN) contents than the MOV-30 and MOV-60 cheeses. Urea-polyacrylamide gel electrophoresis (urea-PAGE) of the pH 4.6-soluble N extract showed that the MOV-60 cheeses had fewer bands derived from casein (CN) hydrolysis than the LOV-30 or MOV-30 cheeses, despite its having exhibited the highest plasmin (PL) activity levels. Our results suggest that the ventilation regimen is critical in dairy sheep housing for optimizing the hygienic quality of ewe milk and the proteolytic processes occurring in Canestrato Pugliese cheese during ripening.