Abstract The aim of the present thesis was the study of the effects of nutrition on immune system of dairy ewes under different types of stressors. The experiments were divided in four different trials. The first experiment was undertaken to evaluate the potential effects of phytosterols extracted from a microalga, intented to be used in sheep nutrition as feed supplements, on in vitro immunological responses of cells from dairy ewes. The second and the third trials were undertaken in order to evaluate the effects of PUFA supplementation on welfare and immune responses of sheep under heat stress. The second trial evaluated the effects of supplementation with polyunsaturated fatty acids from seaweed and flaxseed on welfare and in vivo immunological profile and HPA-axis activation during exposition of ewes to high ambient temperature. The third experiment studied the effects of supplementation with polyunsaturated fatty acids from seaweed and flaxseed on ex vivo inflammatory profile of cells from dairy ewes under high ambient temperature. The fourth trial evaluated the effects of supplementation with polyunsaturated fatty acids from flaxseed on immunological profile in dairy ewes during post partum. In the first experiment peripheral blood mononuclear cells (PBMC) were isolated from sheep. PBMC were treated to determine cell proliferation and cytokine production with different sterols: ergosterol (E), a mixture of eleven Algae sterols extracted and purified from Dunaliella tertiolecta (Algae Extract, AE), a mixture of ergosterol and 7-dehydroporiferasterol extracted and purified from Dunaliella tertiolecta (Purified Extract, PE). Cytokine production (TNF-α, IL- 6, IL-1β, and IL-10) was evaluated after cell treatment with Concanavalin A (Con A) and lipopolysaccharide (LPS). The mixture of ergosterol and 7-dehydroporiferasterol extracted and purified from Dunaliella tertiolecta showed a suppressive effect on cell proliferation, and a reduction of pro-inflammatory cytokines production. Furthermore, a stimulatory effect on the production of the regulatory cytokine IL-10 was found. The immunosuppressive effect exerted by the mixture of ergosterol and 7-dehydroporiferasterol from Dunaliella tertiolecta was dose-dependent both in suppressing cell proliferation and in stimulating IL-10 production. Present results showed that the immunomodulatory and anti-inflammatory activities were more apparent in the purified extract characterized by the mixture of ergosterol and 7-dehydroporiferasterol, and might depend on the existence of a synergic effect of the structures of the two phytosterols. Furthermore, findings from our study suggest that the purified extract characterized by the mixture of ergosterol and 7-dehydroporiferasterol from Dunaliella tertiolecta could be used to reduce immune reactions resulting from inflammatory diseases in sheep production systems, and could have innovative implications on the modulation of sheep immune system when used as feed supplements. The second experiment was carried out during the summer of 2012: thirty-two Comisana ewes were divided into four groups of eight. The FS group was supplemented with whole flaxseed, the AG group was supplemented with Ascophyllum nodosum, the FS+AG group was supplemented with a combination of flaxseed and Ascophyllum nodosum. The C group acted as a control. At 22 d of the experiment, cortisol concentrations in sheep blood were measured after an injection of adrenocorticotropic hormone (ACTH). Cellular immune response was evaluated by intradermic injection of phytohemagglutinin (PHA) at the beginning, and then at 15 d, and 30 d of the trial. Humoral response to ovalbumin (OVA) was measured at 0 d, 15 d, and 30 d. At 0 d, 15 d, and 30 d of the experiment, blood samples were collected from each ewe for determination of T-helper (Th)1 cytokines production (IL-12, and IFN-γ), and Th2 cytokine production (IL-10, IL-4, IL-13), and for determination of heat shock protein (HSP) 70 and 90 concentrations. Ewes supplemented with flaxseed displayed greater cortisol concentrations and a long lasting cell-mediated immune response compared with the control and other experimental groups. Anti-OVA IgG concentrations rose in all groups throughout the trial, even though at 15 d ewes in the FS+AG group displayed the lowest anti-OVA IgG concentrations. IL-10 level increased in all groups throughout the experiment; the FS+AG group had the lowest IL-13 concentration at 15 d, and 30 d. HSP 70 increased in AG ewes at the end of the experiment, and decreased in FS ewes, while HSP 90 concentration 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. In the third experiment thirty-two Comisana ewes were divided into four groups of eight each. The FS group was supplemented with whole flaxseed, the AG group was supplemented with Ascophyllum nodosum, the FS+AG group was supplemented with a combination of flaxseed and Ascophyllum nodosum. The C group acted as a control. The third experiment was carried out during the summer of 2012. At 15 d of the experiment blood samples were collected in vacuum tubes from the jugular vein of sheep to isolate peripheral blood mononuclear cells (PBMC). Lymphocyte proliferation and production of interleuchin (IL)-6, IL-10, and IFN-γ were tested. PBMC from sheep fed Ascophyllum nodosum (AG) proliferated less and produced less IL-6 than PBMC from FS+AG and C groups. FS+AG cells produced lower levels of IL-10 than C cells, and higher IL-6 than AG and C cells. Results demonstrated that the supplementation with PUFA from different sources in sheep diet can influence their immunological responses under high ambient temperatures depending on the composition of fatty acid supplementation. In particular, synergistic effects of different PUFA, simultaneously administrated in the sheep diet, on activation of inflammation response have been observed. In the fourth experiment twenty parturient Comisana ewes were divided into two groups of ten each. FS ewes were supplemented with whole flaxseed, the C ewes represented the control. Peripheral blood samples from each ewe were collected from the jugular vein of ewes immediately before the first injection of OVA (0 d) and then at 7, 14, 21, 28, and 42 d from the day of parturition. Plasma samples were used to perform ELISAs to evaluate the anti-OVA IgG titers; at 0, 14, and 42d from parturition IL-1β, IL-6, IL-10 and TNF-α concentrations were measured in the plasma samples. The plasma anti-OVA IgG titres measured in this experiment displayed a reduced responsiveness to antigen stimulation both in C and in FS ewes. The production of IL-6 in C ewes registered an increase at parturition and then decreased throughout post partum; in FS ewes the levels of IL-6 registered at 14 d post partum were higher than those of IL-6 in C ewes; the same trend was observed for IL-10 production, which resulted two-fold higher at 14 d post partum in FS ewes than in C ewes. The lower concentration of IL-1β in FS ewes around parturition than in C ewes could be related to PUFA content of flaxseed, while the level of TNF-α in FS ewes at the end of post partum registered a decrease compared with levels of TNF-α in C ewes. This results demonstrated that flaxseed supplementation can contribute to reduce the duration of immune depression during post partum, by altering cytokines production, and improving ewes’ ability to respond to infections.

NUTRITION AND STRESS: A field study on the effects of diet on stress-related responses in sheep / Ciliberti, MARIA GIOVANNA. - (2014 Apr 11). [10.14274/UNIFG/FAIR/331736]

NUTRITION AND STRESS: A field study on the effects of diet on stress-related responses in sheep.

CILIBERTI, MARIA GIOVANNA
2014-04-11

Abstract

Abstract The aim of the present thesis was the study of the effects of nutrition on immune system of dairy ewes under different types of stressors. The experiments were divided in four different trials. The first experiment was undertaken to evaluate the potential effects of phytosterols extracted from a microalga, intented to be used in sheep nutrition as feed supplements, on in vitro immunological responses of cells from dairy ewes. The second and the third trials were undertaken in order to evaluate the effects of PUFA supplementation on welfare and immune responses of sheep under heat stress. The second trial evaluated the effects of supplementation with polyunsaturated fatty acids from seaweed and flaxseed on welfare and in vivo immunological profile and HPA-axis activation during exposition of ewes to high ambient temperature. The third experiment studied the effects of supplementation with polyunsaturated fatty acids from seaweed and flaxseed on ex vivo inflammatory profile of cells from dairy ewes under high ambient temperature. The fourth trial evaluated the effects of supplementation with polyunsaturated fatty acids from flaxseed on immunological profile in dairy ewes during post partum. In the first experiment peripheral blood mononuclear cells (PBMC) were isolated from sheep. PBMC were treated to determine cell proliferation and cytokine production with different sterols: ergosterol (E), a mixture of eleven Algae sterols extracted and purified from Dunaliella tertiolecta (Algae Extract, AE), a mixture of ergosterol and 7-dehydroporiferasterol extracted and purified from Dunaliella tertiolecta (Purified Extract, PE). Cytokine production (TNF-α, IL- 6, IL-1β, and IL-10) was evaluated after cell treatment with Concanavalin A (Con A) and lipopolysaccharide (LPS). The mixture of ergosterol and 7-dehydroporiferasterol extracted and purified from Dunaliella tertiolecta showed a suppressive effect on cell proliferation, and a reduction of pro-inflammatory cytokines production. Furthermore, a stimulatory effect on the production of the regulatory cytokine IL-10 was found. The immunosuppressive effect exerted by the mixture of ergosterol and 7-dehydroporiferasterol from Dunaliella tertiolecta was dose-dependent both in suppressing cell proliferation and in stimulating IL-10 production. Present results showed that the immunomodulatory and anti-inflammatory activities were more apparent in the purified extract characterized by the mixture of ergosterol and 7-dehydroporiferasterol, and might depend on the existence of a synergic effect of the structures of the two phytosterols. Furthermore, findings from our study suggest that the purified extract characterized by the mixture of ergosterol and 7-dehydroporiferasterol from Dunaliella tertiolecta could be used to reduce immune reactions resulting from inflammatory diseases in sheep production systems, and could have innovative implications on the modulation of sheep immune system when used as feed supplements. The second experiment was carried out during the summer of 2012: thirty-two Comisana ewes were divided into four groups of eight. The FS group was supplemented with whole flaxseed, the AG group was supplemented with Ascophyllum nodosum, the FS+AG group was supplemented with a combination of flaxseed and Ascophyllum nodosum. The C group acted as a control. At 22 d of the experiment, cortisol concentrations in sheep blood were measured after an injection of adrenocorticotropic hormone (ACTH). Cellular immune response was evaluated by intradermic injection of phytohemagglutinin (PHA) at the beginning, and then at 15 d, and 30 d of the trial. Humoral response to ovalbumin (OVA) was measured at 0 d, 15 d, and 30 d. At 0 d, 15 d, and 30 d of the experiment, blood samples were collected from each ewe for determination of T-helper (Th)1 cytokines production (IL-12, and IFN-γ), and Th2 cytokine production (IL-10, IL-4, IL-13), and for determination of heat shock protein (HSP) 70 and 90 concentrations. Ewes supplemented with flaxseed displayed greater cortisol concentrations and a long lasting cell-mediated immune response compared with the control and other experimental groups. Anti-OVA IgG concentrations rose in all groups throughout the trial, even though at 15 d ewes in the FS+AG group displayed the lowest anti-OVA IgG concentrations. IL-10 level increased in all groups throughout the experiment; the FS+AG group had the lowest IL-13 concentration at 15 d, and 30 d. HSP 70 increased in AG ewes at the end of the experiment, and decreased in FS ewes, while HSP 90 concentration 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. In the third experiment thirty-two Comisana ewes were divided into four groups of eight each. The FS group was supplemented with whole flaxseed, the AG group was supplemented with Ascophyllum nodosum, the FS+AG group was supplemented with a combination of flaxseed and Ascophyllum nodosum. The C group acted as a control. The third experiment was carried out during the summer of 2012. At 15 d of the experiment blood samples were collected in vacuum tubes from the jugular vein of sheep to isolate peripheral blood mononuclear cells (PBMC). Lymphocyte proliferation and production of interleuchin (IL)-6, IL-10, and IFN-γ were tested. PBMC from sheep fed Ascophyllum nodosum (AG) proliferated less and produced less IL-6 than PBMC from FS+AG and C groups. FS+AG cells produced lower levels of IL-10 than C cells, and higher IL-6 than AG and C cells. Results demonstrated that the supplementation with PUFA from different sources in sheep diet can influence their immunological responses under high ambient temperatures depending on the composition of fatty acid supplementation. In particular, synergistic effects of different PUFA, simultaneously administrated in the sheep diet, on activation of inflammation response have been observed. In the fourth experiment twenty parturient Comisana ewes were divided into two groups of ten each. FS ewes were supplemented with whole flaxseed, the C ewes represented the control. Peripheral blood samples from each ewe were collected from the jugular vein of ewes immediately before the first injection of OVA (0 d) and then at 7, 14, 21, 28, and 42 d from the day of parturition. Plasma samples were used to perform ELISAs to evaluate the anti-OVA IgG titers; at 0, 14, and 42d from parturition IL-1β, IL-6, IL-10 and TNF-α concentrations were measured in the plasma samples. The plasma anti-OVA IgG titres measured in this experiment displayed a reduced responsiveness to antigen stimulation both in C and in FS ewes. The production of IL-6 in C ewes registered an increase at parturition and then decreased throughout post partum; in FS ewes the levels of IL-6 registered at 14 d post partum were higher than those of IL-6 in C ewes; the same trend was observed for IL-10 production, which resulted two-fold higher at 14 d post partum in FS ewes than in C ewes. The lower concentration of IL-1β in FS ewes around parturition than in C ewes could be related to PUFA content of flaxseed, while the level of TNF-α in FS ewes at the end of post partum registered a decrease compared with levels of TNF-α in C ewes. This results demonstrated that flaxseed supplementation can contribute to reduce the duration of immune depression during post partum, by altering cytokines production, and improving ewes’ ability to respond to infections.
11-apr-2014
Heat stress, sheep, phytosterol, PUFA, post partum, cytokines, proliferation.
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