The Effect of Dietary Supplementation with Probiotic and Postbiotic Yeast Products on Ewes Milk Performance and Immune Oxidative Status.

The administration of yeast products as feed additives has been proven to beneficially affect animal productivity through energy, oxidative, and immune status improvement. This study evaluated a combination of Saccharomyces cerevisiae live yeast (LY) with yeast postbiotics (rich in mannan-oligosaccharides (MOS) and beta-glucans) and selenium (Se)-enriched yeast on ewes' milk performance and milk quality, energy and oxidative status, and gene expression related to their immune system during the peripartum period. Ewes were fed a basal diet (BD; F:C = 58:42 prepartum and 41:59 postpartum) including inorganic Se (CON; n = 27), the BD supplemented with a LY product, and inorganic Se (AC; n = 29), as well as the combination of the LY, a product of yeast fraction rich in MOS and beta-glucans, and organic-Se-enriched yeast (ACMAN; n = 26) from 6 weeks prepartum to 6 weeks postpartum. The ß-hydroxybutyric acid concentration in the blood of AC and ACMAN ewes was lower (compared to the CON) in both pre- and postpartum periods (p < 0.010). Postpartum, milk yield was increased in the AC and ACMAN Lacaune ewes (p = 0.001). In addition, the activity of superoxide dismutase (p = 0.037) and total antioxidant capacity (p = 0.034) measured via the 2,2-Azino-bis (3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) method was increased in the blood plasma of the ACMAN postpartum. Higher ABTS values were also found (p = 0.021), while protein carbonyls were reduced (p = 0.023) in the milk of the treated groups. The relative transcript levels of CCL5 and IL6 were downregulated in the monocytes (p = 0.007 and p = 0.026 respectively), and those of NFKB were downregulated in the neutrophils of the ACMAN-fed ewes postpartum (p = 0.020). The dietary supplementation of ewes with yeast postbiotics rich in MOS and beta-glucans, and organic Se, improved energy status, milk yield and some milk constituents, and oxidative status, with simultaneous suppression of mRNA levels of proinflammatory genes during the peripartum period.

Schizochytrium spp. Dietary Supplementation Modulates Immune-Oxidative Transcriptional Signatures in Monocytes and Neutrophils of Dairy Goats.

The high propensity of dietary polyunsaturated fatty acids (PUFA) to oxidation can induce a cascade of cellular immune-oxidative imbalances. On the other hand, PUFA, namely docosapentaenoic acid (ω6-DPA) and docosahexaenoic acid (DHA) can exert immunomodulatory effects by suppressing a pro-inflammatory response. Thus, the objective of this study was to investigate the effect of dietary supplementation with Schizochytrium spp. levels, rich in both ω6-DPA and DHA on the transcriptional profiling of genes involved in oxidative homeostasis and innate immunity of dairy goats' monocytes and neutrophils. Twenty-four dairy goats were divided into four homogeneous sub-groups; the diet of the control group (CON) had no Schizochytrium spp. while those of the treated groups were supplemented daily with 20 (ALG20), 40 (ALG40), and 60 (ALG60) g/goat/day. The mRNA levels of MGST1 in neutrophils were downregulated (p = 0.037), while in monocytes, SOD2 and SOD3 were downregulated (p = 0.010 and p = 0.044, respectively) in ALG60 compared to the CON group. GPX2 mRNA levels were downregulated (p = 0.036) in ALG20 and ALG60 compared to the CON group in neutrophils. NOX1 was upregulated (p = 0.043) in the neutrophiles of ALG60-goats. NOX2 was upregulated (p = 0.042) in the monocytes of ALG40-fed goats, while higher (p = 0.045) levels were also found in the ALG60 group in neutrophils. The mRNA levels of COX2 were downregulated (p = 0.035) in monocytes of the ALG40 and ALG60 groups. The mRNA levels of PTGER2 were also downregulated (p = 0.004) in monocytes of Schizochytrium-fed goats, while in neutrophils, significant downregulation (p = 0.024) was only found for ALG60 compared to the CON group. ALOX5AP mRNA levels were significantly decreased (p = 0.033) in ALG60 compared to the CON group in monocytes. LTA4H mRNA levels were increased (p = 0.015) in ALG60 compared to ALG20 and ALG40 groups in monocytes, while in neutrophils, a significant downregulation (p = 0.028) was observed in ALG20 compared to the CON group. The inclusion of more than 20 g Schizochytrium spp./day in goats' diet induced imbalances in mechanisms that regulate the antioxidant system, while downregulated the expression of pro-inflammatory pathways in monocytes and neutrophils.

The Effect of Dietary Inclusion of Microalgae Schizochytrium spp. on Ewes' Milk Quality and Oxidative Status.

An unprecedented challenge for nutritionists arises during the 21st century in order to produce highly nutritious and functional food which promotes human health. Polyunsaturated fatty acids (PUFA) that are highly contained in microalgae have broadly been confirmed for preventing cardiovascular diseases and regulating immune-oxidative status. However, their optimum dietary inclusion level needs to be defined since PUFA are prone to oxidation. For this purpose, 24 cross-bred dairy ewes, were separated into four groups (n = 6) and were fed with different levels of microalgae Schizochytrium spp. [0 (CON, no microalgae), 20 (SC20), 30 (SC30) and 40 (SC40) g/ewe/day] for 60 days. The results showed that although the production parameters were not impaired, milk fat content was decreased in medium and high-level supplemented groups while protein content was suppressed only for the medium one. Concerning the fatty acids (FA) profile, the proportions of C14:0, trans C18:1, trans-11 C18:1, cis-9, trans-11 C18:2, trans-10, cis-12 C18:2, C20:5 (EPA), C22:5n-6 (DPA), C22:6n-3 (DHA), the total ω3 FA and PUFA were significantly increased, while those of C18:0, cis-9 C18:1 and C18:2n-6c were decreased in the milk of treated ewes. Additionally, in the S40 group an oxidative response was induced, observed by the increased malondialdehyde (MDA) levels in milk and blood plasma. In conclusion, the dietary inclusion of 20 g Schizochytrium spp./ewe/day, improves milks' fatty acid profile and seems to be a promising way for producing ω3 fatty acid-enriched dairy products.

Long-term administration of a commercial supplement enriched with bioactive compounds does not affect feed intake, health status, and growth performances in beef cattle.

Feed additives including natural bioactive compounds (BCs) in combination with vitamin E (VitE) and organic Se could mitigate animal stress associated with intensive livestock farming due to their anti-inflammatory and antioxidant properties. Yeast and yeast derivate are included in feed additives as probiotic products and digestion promoters. Scutellaria baicalensis is a source of bioactive compounds and has been tested in monogastrics, exhibiting many immunostimulating and hepato-protective activities. However, the literature lacks information regarding S. baicalensis effects on beef cattle performance and health status. The aim of the present study was to evaluate the impact on beef cattle's feed intake, health and oxidative status, and growth performances of the inclusion of a commercial supplement (CS) containing VitE, organic Se, yeast derivate, and S. baicalensis extract during the fattening and finishing period. A total of 143 Charolaise male cattle were allotted into 12 pens of 11-12 animals each and assigned to a control ( 463.9 ± 21.48 body weight - BW) or a treated ( 469.8 ± 17.91  BW) group. Each group included two replicates of three pens. The treated groups were supplemented with 20  g CS animal - 1 d - 1 . Feed intake was measured monthly on a pen base during two consecutive days. Total mixed ration and fecal samples were collected at three time points (monthly, from November to February) and pooled by replicate for the analyses to monitor digestibility. Blood samples were individually collected at the beginning and at the end of the trial for oxidative status and metabolic profile determination. Final BW and carcass weight were individually recorded to calculate average daily gain, feed conversion ratio, and carcass yield. Similar feed digestibility between groups were observed during the whole experiment. Feed intake, growth performances, final body weight, average daily gain, feed conversion rate, oxidative status, and metabolic profile were not affected by the dietary inclusion of the tested CS indicating no detrimental effect of the treatment. Different doses of this product should be tested in the future in order to provide a more complete report on the product efficacy.

Effect of a Carotenoid Extract from Citrus reticulata By-Products on the Immune-Oxidative Status of Broilers.

Although carotenoids generally possess antimicrobial and antioxidant properties, the in vivo synergistic action of carotenoid blends derived from plant-based by-products has not been thoroughly studied. Therefore, the carotenoid characterization and antimicrobial potential of Citrus reticulata extract as well as the impact of this carotenoid-rich extract (CCE) dietary supplementation on the performance, meat quality, and immune-oxidative status of broiler chickens were determined. One hundred and twenty one-day-old hatched chicks (Ross 308) were allocated to two dietary groups, with four replicate pens of 15 birds each. Birds were fed either a basal diet (CON) or the basal diet supplemented with 0.1% CCE (25 mg carotenoid extract included in 1 g of soluble starch) for 42 d. ß-Cryptoxanthin, ß-Carotene, Zeaxanthin, and Lutein were the prevailing carotenoid compounds in the Citrus reticulata extract. The CCE feed additive exerted inhibitory properties against both Gram-positive (Staphylococcus aureus) and negative (Klebsiella oxytoca, Escherichia coli, and Salmonella typhimurium) bacteria. Both the broiler performance and meat quality did not substantially differ, while the breast muscle malondialdehyde (MDA) concentration tended to decrease (p = 0.070) in the CCE-fed broilers. The inclusion of CCE decreased the alanine aminotransferase and MDA concentration, and the activity of glutathione peroxidase, while the activity of superoxide dismutase was increased in the blood. Catalase and NADPH oxidase 2 relative transcript levels were significantly downregulated in the livers of the CCE-fed broilers. Additionally, Interleukin 1ß and tumor necrosis factor (TNF) relative transcript levels were downregulated in the livers of the CCE- fed broilers, while TNF and interferon γ (IFNG) tended to decrease in the spleens and bursa of Fabricius, respectively. The present study provided new insights regarding the beneficial properties of carotenoids contained in Citrus reticulata in broilers' immune-oxidative status. These promising outcomes could be the basis for further research under field conditions.

Effects of PUFA-Rich Dietary Strategies on Ruminants' Mammary Gland Gene Network: A Nutrigenomics Review.

Although the inclusion of polyunsaturated fatty acids (PUFAs) in ruminants' diets appears to be a well-documented strategy to enrich milk with PUFAs, several gene networks that regulate milk synthesis and mammary gland homeostasis could be impaired. The objective of this literature review is to assess the effects of nutritional strategies focused on enriching milk with PUFAs on gene networks regulating mammary gland function and lipogenesis, as well as the impact of feed additives and bioactive compounds with prominent antioxidant potential on immune-oxidative transcriptional profiling, as a part of mammary gland homeostasis and health. The findings support the conclusion that PUFAs' inclusion in ruminants' diets more strongly downregulate the stearoyl-CoA desaturase (SCD) gene compared to other key genes involved in de novo fatty acid synthesis in the mammary gland. Additionally, it was revealed that seed oils rich in linoleic and linolenic acids have no such strong impact on networks that regulate lipogenic homeostasis compared to marine oils rich in eicosapentaenoic and docosahexaenoic acids. Furthermore, ample evidence supports that cows and sheep are more prone to the suppression of lipogenesis pathways compared to goats under the impact of dietary marine PUFAs. On the other hand, the inclusion of feed additives and bioactive compounds with prominent antioxidant potential in ruminants' diets can strengthen mammary gland immune-oxidative status. Considering that PUFA's high propensity to oxidation can induce a cascade of pro-oxidant incidences, the simultaneous supplementation of antioxidant compounds and especially polyphenols may alleviate any side effects caused by PUFA overload in the mammary gland. In conclusion, future studies should deeply investigate the effects of PUFAs on mammary gland gene networks in an effort to holistically understand their impact on both milk fat depression syndrome and homeostatic disturbance.

Schizochytrium sp. Dietary supplementation modify Toll-like receptor 4 (TLR4) transcriptional regulation in monocytes and neutrophils of dairy goats.

Animals protect themselves against pathogens or abiotic factors by innate or adaptive mechanisms. Long-chain polyunsaturated fatty acids (ω3) of microalgae modify both human and mice' immune systems resulting in a beneficial balance between pro-inflammatory and anti-inflammatory pathways. However, scarce information exists on their impact on lactating animals' immunity. The objective of this study was to investigate the impact of dietary inclusion of Schizochytrium sp. (rich in docosapentaenoic and docosahexaenoic acid), on the expression of several genes involved in the innate immunity of goats. Twenty-four dairy goats were divided into four homogeneous sub-groups (n = 6). All goats were fed individually with alfalfa hay and concentrate. The concentrate of the control group (CON) had no microalgae while those of the treated groups were supplemented daily with 20 (ALG20), 40 (ALG40), and 60 (ALG60) g Schizochytrium sp. Monocytes and neutrophils were isolated from goats' blood in the 20th, 40th, and 60th days from the beginning of the experimental period. The relative transcript levels of TLR4, MYD88, MAPK, IRF3, IFNG, and pro-inflammatory cytokines (IL1B, IL2, IL8, TNF), and chemokines (CCL5 and CXCL16) were decreased in monocytes of microalgae treated goats compared to the CON. In contrast, MAPK and IL1B relative transcript levels were increased in neutrophils of ALG40 and ALG60 groups. In conclusion, the supplementation of goats' diet with 20 g Schizochytrium sp. resulted in a downregulation of the pro-inflammatory transcriptions, and following further research could be considered as a sustainable alternative strategy to improve immune function.

Effects of Inclusion of Schizochytrium spp. and Forage-to-Concentrate Ratios on Goats' Milk Quality and Oxidative Status.

Although the dietary inclusion level of polyunsaturated fatty acids (PUFA) and the forage: concentrate (F:C) ratio affect milk quality, their interaction has not been broadly studied. To address such gaps and limitations a two-phase trial using twenty-two dairy goats was carried out. During the first phase, both groups (20 HF n = 11; high forage and 20 HG n = 11; high grain) were supplemented with 20 g Schizochytrium spp./goat/day. The 20 HF group consumed a diet with F:C ratio 60:40 and the 20 HG-diet consisted of F:C = 40:60. In the second phase, the supplementation level of Schizochytrium spp. was increased to 40 g/day/goat while the F:C ratio between the two groups were remained identical (40 HF n = 11; high forage and 40 HG n = 11; high grain). Neither the Schizochytrium spp. supplementation levels (20 vs. 40) nor the F:C ratio (60:40 vs. 40:60) affected milk performance. The high microalgae level (40 g) in combination with high grain diet (40 HG) modified the proportions of docosahexaenoic acid (DHA), docosapentaenoic acid (DPA), and conjugated linoleic acid (CLA) and the ω3/ω6 ratio in milk, to a beneficial manner according to human health recommendation guidelines. However, the highest inclusion level of Schizochytrium spp. (40 g) and foremost in combination with the high grain diets (40 HG) induced an oxidative response as observed by the increased protein carbonyls (CP) and malondialdehyde (MDA) levels in milk and blood plasma indicating severe limitations for a long-term, on-farm application. In conclusion, the supplementation with 20 g Schizochytrium spp. and high forage diet (60:40) appears to be an ideal formula to enrich dairy products with essential biomolecules for human health without adversely affect milk oxidative stability.

Effects of Supplementing Rumen-Protected Methionine and Lysine on Milk Performance and Oxidative Status of Dairy Ewes.

There is limited information on the impact of dietary supplementation with separate rumen-protected (RP) amino acids (AA), or with their combination, on ewes' oxidative status. Sixty ewes were divided into five groups; C: basal diet (control); M: basal diet + 6 g/ewe RP methionine; L: basal diet + 5 g/ewe RP lysine; LML: basal diet + 6 g methionine and 5 g lysine/ewe; and HML: basal diet + 12 g methionine + 5 g lysine/ewe. Milk's fat content increased in RP-AA fed ewes, while that of protein in M and L only. In blood plasma, the malondialdehyde (MDA) content was reduced in the M, LML, and HML compared to C-fed ewes. An increase in glutathione transferase activity in the blood plasma of the M and LML compared to the C and HML-fed ewes were found. In milk, lower values of the ferric reducing ability of plasma (FRAP) in the LML and HML-fed ewes and of 2,2'-Azino-bis 3-ethylbenzthiazoline-6-sulfonic acid (ABTS) in LML only, were found. Lysine increased milk's FRAP values and MDA content. Both L and HML diets increased milk's protein carbonyls content. Methionine improves the organism's oxidative status, without adversely affecting milk's oxidative stability. Lysine dietary inclusion affects negatively the oxidative stability of milk.

The impact of rumen-protected amino acids on the expression of key- genes involved in the innate immunity of dairy sheep.

Rumen protected amino acids inclusion in ewes' diets has been proposed to enhance their innate immunity. The objective of this work was to determine the impact of dietary supplementation with rumen-protected methionine or lysine, as well as with a combination of these amino acids in two different ratios, on the expression of selected key-genes (NLRs, MyD88, TRIF, MAPK-1, IRF-3, JunD, TRAF-3, IRF-5, IL-1α, IL-10, IKK-α, STAT-3 and HO-1). Thus, sixty Chios dairy ewes (Ovis aries) were assigned to one of the following five dietary treatments (12 animals/ treatment): A: basal diet consist of concentrates, wheat straw and alfalfa hay (control group); B: basal diet +6.0 g/head rumen-protected methionine; C: basal diet + 5.0 g/head rumen-protected lysine; D: basal diet +6.0 g/head rumen-protected methionine + 5.0 g/head rumen-protected lysine and E: basal diet +12.0 g/head rumen-protected methionine + 5.0 g/head rumen-protected lysine. The results revealed a significant downregulation of relative transcript level of the IL-1α gene in the neutrophils of C and in monocytes of D ewes compared with the control. Significantly lower mRNA transcript accumulation was also observed for the MyD88 gene in the neutrophils of ewes fed with lysine only (C). The mRNA relative expression levels of JunD gene were highly induced in the monocytes, while those of IL-10 and HO-1 genes were declined in the neutrophils of ewes fed with the C and D diets compared with the control. Lower transcript levels of STAT-3 gene were observed in the neutrophils of ewes fed with either C or with E diets in comparison with the control. In conclusion, our results suggest that the dietary supplementation of ewes with rumen-protected amino acids, down regulate the expression of some genes involved in the pro-inflammatory signalling.

The effects of dietary supplementation with rumen-protected amino acids on the expression of several genes involved in the immune system of dairy sheep.

Amino acids might be a tool to transform animals from a pro- to an anti-inflammatory phenotype through the downregulation of several genes (TLR-4, NF-κB, TNFa, IL-1ß, IL-2, IL-6, IL-8, CCL-5 and CXCL-16) whose expression increases during inflammation. To examine this possibility, each of sixty Chios dairy sheep was assigned to one of the following five dietary treatments: A: basal diet (control group); B: basal diet +6.0 g/head rumen-protected methionine (MetaSmart™ ); C: basal diet +5.0 g/head rumen-protected lysine (LysiGEM™ ); D: basal diet +6.0 g/head MetaSmart™  + 5.0 g/head LysiGEM™ ; and E: basal diet +12.0 g/head MetaSmart™  + 5.0 g/head LysiGEM. The results showed a significant downregulation in the expression of the TLR-4 gene in both the macrophages and the neutrophils of ewes fed rumen-protected amino acids. Significantly lower mRNA transcript accumulation was also observed for the TNFa, IL-1ß and CXCL-16 genes in the macrophages and for the IL-1ß gene in the neutrophils of ewes supplemented with amino acids. The ewes that received dietary supplementation with rumen-protected lysine alone (C) had significantly lower CCL-5 transcript levels in their macrophages than the ewes fed the other supplemented diets. Diet D enhanced the mRNA expression of the IL-2 gene in ewe neutrophils. Negative correlations were found between: a. TLR-4, TNFa, IL-1ß and CXCL-16 gene expression in macrophages and the milk fat and total solids content; b. CCL-5 gene expression in neutrophils and the milk yield and FCM(6%) ; and c. CXCL-16 gene expression and the milk protein content. Moreover, positive correlations were found between the BHBA concentration and the expression of the TLR-4 and CXCL-16 genes in macrophages. In conclusion, the rumen-protected amino acids improved sheep metabolism (as indicated by reduced blood BHBA and urea concentrations), milk chemical composition and immune system function.