The objective of this study was to determine the effect of cooling upon calving in alleviating the adverse effects of heat stress in Holstein lactating cows. Production performance, indicators of metabolic status, immune response, and biomarkers of oxidative stress were measured. Based on mature equivalent milk production, parity, and calving date, 46 multiparous lactating cows were allotted to groups of equal sizes (n = 23); heat stressed (HS; BW = 658 ± 28 kg [mean ± SD]; BCS = 2.7 ± 0.18; parity = 3 ± 0.12) and cooled (CL; BW = 668 ± 23 kg; BCS = 2.8 ± 0.14; parity = 3 ± 0.25). Cows were housed in sand-bedded individual stalls equipped with misters and fans which were on from 1000 to 1800 hours for CL group. DMI and milk yield were measured from calving for 7 wk. Body condition score and BW were recorded weekly. Blood samples were collected weekly to measure the metabolic and antioxidant status, inflammatory cytokines, and immunoglobulins. Rectal temperature was measured daily at 1400 hour. Mean daily maximum temperature, minimum relative humidity, and maximum temperature–humidity index was 37.0 °C, 31.9%, and 83.4 for HS and 27.3 °C, 44.9%, and 75.7 for CL, respectively. Heat-stressed cows exhibited greater rectal temperature (39.8 vs. 39.1 °C) and lower feed intake (19.8 vs. 21.3 kg/d) relative to CL cows. Milk yield, including raw (31.2 vs. 38.6 kg/d) and fat- and protein-corrected (32.1 vs. 35.7 kg/d) milk, was lower in HS vs. CL cows, respectively. The percentages of milk protein (3.25 vs. 3.06), lactose (4.73 vs. 4.58), and solids-not-fat (8.63 vs. 8.38) but not milk fat (4.31 vs. 3.59) were higher in HS cows than in CL cows, respectively. Somatic cell score was greater in HS cows as compared with CL cows. Cooled cows lost less body condition as compared with HS cows. Blood plasma concentrations of glucose, non-esterified fatty acids, and β-hydroxybutyric acid were lower in HS cows. Blood plasma concentrations of malondialdehyde (2.13 vs. 1.84 nmol/mL), reactive oxygen species (579 vs. 561 U/mL), and total antioxidant capacity (4.49 vs. 4.06 U/mL) were greater in HS cows than in CL cows. Blood plasma concentrations of the inflammatory cytokines (tumor necrosis factor-α, interleukin-1α, and interleukin-2) and immunoglobulins (IgA, IgM, and IgG) were lower in HS cows than in CL cows. These findings demonstrated that cooling dairy cows during the early postpartum improved the production performance, indicators of metabolic status, immune response, and antioxidant capacity.

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 24h and 96h 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 (24h 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 (96h 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.

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.

The objective of this experiment was to study the effects of the unsaponified fraction (UP), the acetylated unsaponified fraction (AUP), and the total lipid fraction (TL) extracted and purified from Chlorella sorokiniana (CS) on the proliferation and cytokine profile of sheep peripheral blood mononuclear cells (PBMCs). Cells were cultured with 0.4 mg/mL and 0.8 mg/mL concentrations of each extract (UP, AUP, and TL fractions) and activated with 5 μg/mL concanavalin A (ConA) and 1 μg/mL lipopolysaccharide (LPS) at 37 °C for 24 h. PBMCs cultured with ConA and LPS represented the stimulated cells (SC), and PBMCs without ConA and LPS represented the unstimulated cells (USC). Cell-free supernatants were collected to determine IL-10, IL-1β, and IL-6 secretions; on cells, measurement of proliferation was performed. All the extracts tested significantly decreased the cell proliferation; in particular, the UP fraction at 0.4 mg/mL showed the lowest proliferative response. Furthermore, at 0.8 mg/mL, the UP fraction enhanced IL-10 secretion. On the contrary, the TL fraction at 0.4 mg/mL induced an increase in IL-10, IL-6, and, to a lesser extent, IL-1β secretions by cells. The AUP fraction did not change cytokine secretion. The results demonstrated that CS extracts could be useful ingredients in animal feed in order to minimize the use of antibiotics by modulating cell proliferation and cytokine response.

Immune competence of the ewe mammary gland was investigated by monitoring the leukocyte differential count, cytokine pattern, and endogenous proteolytic enzymes in milk samples with different somatic cell counts (SCC) and pathogenic bacteria. Furthermore, the leukocyte differential count and T-lymphocyte populations were evaluated in ewe blood. A total of 1,500 individual milk samples were randomly selected from the pool of the samples collected during sampling and grouped into 5 classes of 300 samples each, on the basis of SCC. Classes were 2,000,000 cells/mL. Microbiological analyses of ewe milk were conducted to detect mastitis-related pathogens. Sheep whose udders were without clinical abnormalities, and whose milk was apparently normal but with at least 10(3)cfu/mL of the same pathogen were considered to have subclinical mastitis and therefore defined as infected. Polymorphonuclear neutrophilic leukocytes (PMNL) and macrophages increased with SCC, whereas lymphocytes decreased. Milk samples with SCC >1,000,000 cells/mL showed differences in leukocyte populations between uninfected and infected ewes, with higher percentages of PMNL and macrophages and lower percentages of lymphocytes in infected animals. Nonviable PMNL levels were the highest in ewe milk samples with SCC 500,000 cells/mL, nonviable PMNL were higher in uninfected ewes than in infected ones. In infected animals giving milk with SCC >1,000,000 cells/mL, a higher CD4(+)/CD8(+) ratio was observed, suggesting that the presence of pathogens induced an activation of both CD4(+) and CD8(+). The levels of tumor necrosis factor-α and IL-12 were higher in infected than uninfected ewes, irrespective of SCC. Plasmin activity increased along with SCC and was always higher in infected than uninfected animals; cathepsin D increased starting from 1,001,000 cells/mL in milk samples from noninfected ewes and starting from 301,000 cells/mL in milk samples from infected animals. The associations between somatic cells, cytokines, endogenous proteolytic enzymes, and pathogenic bacteria can be used to better understand the pathogenesis of subclinical mastitis in ewes and the effect on the immune response of ewe mammary gland.