Milk quality of Italian Mediterranean buffalo as affected by temperature-humidity index during late spring and summer.

A total of 18 Italian Mediterranean buffalo cows were subjected to fortnightly milk sampling from May to July 2022. Air temperature and relative humidity were monitored throughout the trial. Temperature-humidity index (THI) was calculated and ranged from 69 to 79, so that 3 classes were set to study the effect of different THI on milk quality: THI less than 72 (THI<72), THI ranging from 72 to 76 (THI72-76), and THI over 76 (THI>76). Individual milk samples from buffalo cows were analyzed for milk composition and coagulation traits, fatty acid and amino acid composition, and enzymatic and mineral profiles. The THI>76 class registered higher lactose content and poorer milk clot characteristics in comparison with THI<72. Exposure to a THI over 76 resulted in increased SFA and short-chain fatty acid contents in milk, as well as in the highest atherogenic and thrombogenic index values and the lowest essential amino acid content. Moreover, the lowest values of calcium, copper, and selenium contents were found in the milk of buffalo cows exposed to a THI over 72. Results confirm that Italian Mediterranean buffalo express a good level of thermo-tolerance, even though exposure to a daily THI over 76 has a deleterious effect on some nutritional and technological properties of milk.

Green extraction of bioactive compounds from wine lees and their bio-responses on immune modulation using in vitro sheep model.

The objective of this study was to apply microwave-assisted extraction using green solvents starting from 3 different wine (white, rosé, and red) lees and to test their bio-response on sheep peripheral blood mononuclear cells proliferation, Bax/Bcl-2 ratio, and cytokines secretion. Wine lees (WL) of local organic farming from white wine, produced with Trebbiano cultivar, rosé and red wine, made with Nero di Troia cultivar, were collected. The WL were subjected to microwave-assisted extraction using 2 green solvents (water and ethanol) in 3 different combinations (water; water/ethanol 1:1 vol/vol; ethanol) with a dry matter-to-solvent ratio of 1:40 (wt/vol) at 4 temperature levels: 50, 100, 150, and 200°C. Sodium carbonate Na2CO3 (2 mmol/g of dry weight of lees) was used for increasing the polyphenol extraction yield. A total number of 6 extracts, 2 for each kind of WL investigated, according to their total phenolic content and in vitro antioxidant capacity, were selected to be tested on sheep peripheral blood mononuclear cells, as an animal model. All the WL extracts demonstrated a strong antiproliferative action. On the contrary, the cytokines' profile was mainly dependent on the different winemaking derived WL and the extraction solvent combination procedures. Red WL extract obtained by a combination of water/Na2CO3 and tested at 0.8 mg/mL, resulted in an increase of both IL-6 secretion and Bax/Bcl-2 ratio. Data from the present study demonstrated that WL extracts derived from different winemaking and solvent extraction could have a bimodal action on control of inflammatory mediated damage and highlighted the importance for further studies aimed at applying the biorefinery process on by-products to increase their economic value and exploit new derived bioactive compound.

Potential benefits of early-life supplementation of liquid feed with fennel (Foeniculum vulgare) seeds or oregano (Origanum vulgare) leaves on growth, health, and blood metabolites in Holstein dairy calves.

This study was performed to determine the early-life (first month of age) supplementation of liquid feed with fennel seed powder (FSP) or oregano leaf powder (OLP) on growth performance, health, and blood biochemical attributes in preweaning dairy calves. Holstein female calves (n = 57; 1 d of age; 34.1 ± 0.97 kg of BW; mean ± SE) were assigned randomly to receive liquid feed (colostrum and milk) with no added herbal plants (CON) or supplemented with FSP (3 g/d) or OLP (30 g/d) during the first month of age. The calves received pooled colostrum (4.5 kg/d on the first 2 d of life; total solids = 25.0% ± 1.24; mean ± SD) and then pooled waste milk (6 kg/d from d 3 to 44, 5 kg/d from d 45 to 46, 4 kg/d from d 47 to 48, and 3 kg/d from d 49 to 50 of the trial; total solids = 12.54% ± 0.50) to ensure they receive same mixed liquid feed daily. The calves had unlimited access to the starter feed and fresh water and remained in the study until weaning on d 51 of age. The average mean temperature-humidity index was 70.1 units (ranging between 61.9 to 78.2) during the experiment, indicating a borderline degree of environmental heat-load. The amount of starter feed offered and refused was recorded daily. The calves were weighed immediately after birth and every 10 d thereafter, before the morning feeding. Jugular blood samples were taken immediately before and 24 h after colostrum feeding, at first month of age, and at weaning to quantify serum concentrations of glucose, urea N, cholesterol, triglycerides, total proteins, albumin, globulin, aspartate transferase, alanine transferase, total antioxidant status, and malondialdehyde. Health checks including rectal temperature, general appearance (on a 1-5 score system), fecal score (on a 1-5 score system), and bovine respiratory disease (BRD; scored using the University of Wisconsin Calf Health Chart) were performed daily, by a veterinarian who was unaware of the calf treatment allocations, for all calves over the study period. A repeated-measures ANOVA was used to compare growth performance and blood metabolites among treatment groups, and a logistic regression model using a binomial distribution (PROC GLIMMIX, SAS v. 9.4, SAS Institute Inc.) was used to assess the chance of elevated rectal temperature (≥39.4°C), general appearance (≥2), diarrhea (≥3), and BRD. A Poisson regression model (PROC GENMOD) was also used to test group differences in the experience of days with elevated rectal temperature and general appearance, and frequency and duration of diarrhea or BRD. Total nutrient intake (DM, CP, and ME, but not ether extract) originating from milk and starter feed was greater in OLP- (but not FSP-) supplemented calves compared with CON group, being partially associated with difference in milk refusal. Calves receiving FSP and OLP had greater average daily gain, BW gain, skeletal gain (withers height or heart girth, respectively), and feed efficiency compared with CON animals with no difference between FSP and OLP. Rectal temperature was lower in FSP- (but not OLP-) supplemented calves compared with CON animals. The CON animals had a greater chance of experiencing higher rectal temperature (≥39.4°C; odds ratio = 1.55 and confidence interval = 1.12-2.15 and odds ratio = 1.33 and confidence interval = 0.92-1.90, respectively, compared with FSP and OLP) and general appearance (≥2; odds ratio = 1.99 and confidence interval = 1.45-2.74 and odds ratio = 1.45 and confidence interval = 1.03-2.05), and diarrhea (odds ratio = 1.47 and confidence interval = 1.11-1.97 and odds ratio = 1.49 and confidence interval = 1.07-2.08) compared with those receiving FSP or OLP; with the chance of BRD being lower in FSP- (but not OLP-) supplemented versus CON animals (odds ratio = 1.59 and confidence interval = 1.13-2.23). As compared with OLP and CON groups, FSP treatment resulted in shorter days with elevated rectal temperature and general appearance. Supplementing FSP or OLP decreased the duration of diarrhea and BRD but not their frequency compared with CON. Duration of diarrhea was not different between FSP- or OLP-supplemented calves but calves supplemented with FSP had shorter days with BRD compared with OLP-supplemented calves. Of the blood constituents measured on d 30, concentration of aspartate transferase was higher in FSP- (but not CON) versus OLP-supplemented calves, indicating a transient liver tissue damage or dysfunction which was subsequently ameliorated. At weaning, blood concentration of triglycerides was higher in FSP and OLP groups compared with CON group. Supplementation of the liquid feed with FSP or OLP, especially FSP, had beneficial effects on calf growth performance and health. Further experiments are warranted for optimizing the dosage and duration of feeding FSP or OLP as feed additives for dairy calves.

Evaluation of natural plant extracts as antioxidants in a bovine in vitro model of oxidative stress.

Oxidative stress contributes to many inflammatory-based diseases of dairy cattle especially during periods of increased metabolic activity such as around calving. Endothelial cells play a key role in maintaining normal inflammatory responses, but they are especially susceptible to macromolecule damage during times of oxidative stress. Therefore, bovine aortic endothelial cells (BAEC) were used to study the effect of natural tannin-based extracts on oxidative stress that may improve health and well-being of cattle. Tannins are secondary metabolites in plants with potent antioxidant activity that have been used as natural feed additives for food-producing animals. However, there is little information on how tannin-rich plant extracts may affect oxidative stress in dairy cattle. The objective of this study was to evaluate the antioxidant effect of pomegranate (Punica granatum; PMG), tara (Caesalpinia spinosa; TA), chestnut (Castanea sativa; CH), and gambier (Uncaria gambir; GM) natural extracts using an in vitro BAEC model of oxidative stress. Natural extracts were tested at a concentration of 80 µg/mL. Viability, apoptosis, intracellular reactive oxygen species, and isoprostanes were determined on cultured BAEC treated with different plant natural extracts. No changes in cell viability was detected following PMG and GM treatments. In contrast, there was a 30% reduction of BAEC viability following treatment with CH or TA extracts. Intracellular reactive oxygen species production was significantly less abundant in cells treated with natural extracts than with the lipopolysaccharide control. Moreover, antioxidant activity varied according to the tested extract, showing a reduction of 63, 45, 51, and 27% in PMG, GM, CH, and TA, respectively. The formation of isoprostanes as a consequence of lipid peroxidation after induction of oxidative stress also were significantly decreased in PMG-treated cells when compared with the untreated cells. Theses findings suggest that PMG extract has the potential to mitigate oxidative stress without detrimental effects on cell viability. Further in vitro and in vivo research is warranted to explore the antioxidant potential of PMG extract as a dietary supplement to control oxidative stress in dairy cattle.

Novel milk-juice beverage with fermented sheep milk and strawberry (Fragaria × ananassa): Nutritional and functional characterization.

This study was aimed at developing a new functional fermented beverage manufactured with semi-skimmed sheep milk and strawberry pulp (Fragaria × ananassa Duch.) and commercial prebiotic ingredients. We also compared the performance of the yogurt starter cultures and a Lactobacillus plantarum strain (CECT_8328) with potential probiotic properties. We assessed the nutritional profile, bioactivity compounds, viability of lactic acid bacteria during storage, and survival of L. plantarum after in vitro simulated digestion during the storage period. The lactic acid bacteria were viable throughout the storage period, but only L. plantarum maintained good viability after simulated digestion. Nevertheless, neither inulin nor potato starch increased bacterial viability. The fermented semi-skimmed sheep milk strawberry beverages we developed are good sources of minerals and proteins.

Glucocorticoid effects on sheep peripheral blood mononuclear cell proliferation and cytokine production under in vitro hyperthermia.

The present experiment aimed at understanding the effects of cortisol levels on sheep peripheral blood mononuclear cell (PBMC) proliferation and cytokine production during hyperthermia. To mimic stress related to the exposition of high ambient temperatures, PBMC were cultured at 43°C for 12 h, and subsequently at 39°C for additional 12 h. Cells in normothermia were cultured at 39°C for 24 h. Phytohemagglutinin-stimulated PBMC were cultured with different cortisol levels: 0 ng/mL; 100 ng/mL, representing the physiological cortisol concentration simulating stress condition (Cort100); and 1,000 ng/mL, representing the hyperactivated hypothalamic-pituitary-adrenal axis (Cort1000). Phytohemagglutinin-stimulated PBMC with 0 ng/mL of cortisol concentration represented the positive control, whereas nonstimulated PBMC without cortisol represented the negative control (NC). The free cell supernatants were collected for the determination of IL-6, IL-1ß, and IL-10 by ELISA. Bromodeoxyuridine assay was performed on cells to determine cell proliferation. Exposition to hyperthermia negatively affected cell proliferation, IL-6, IL-1ß, and IL-10 concentrations in cell supernatants. The interaction of hyperthermia and cortisol level affected both cell proliferation and IL-10 production. Both PBMC proliferation and IL-10 production in positive control, Cort100, and Cort1000 decreased at 43°C as compared with 39°C NC. On average, the Cort100 treatment displayed higher concentrations of IL-6 than NC. The present experiment demonstrated that the action of cortisol concentration simulating stress condition on cell proliferation and cytokine production was a permissive/stimulatory action during normothermia, whereas it was a suppressive action during hyperthermia. These data confirmed that cortisol concentration simulating stress condition could have a role in the immune system of sheep via mediating cellular homeostasis in the condition of hyperthermia. The negative effects of hyperthermia on sheep immune responses were apparent when performing an immunological challenge.

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 [αS1-casein (CN), αS2-CN, κ-CN, ß-CN, and a mix of α-lactalbumin (α-LA) and ß-lactoglobulin (ß-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-1ß was studied in cultured PBMC incubated with αS1-CN, αS2-CN, κ-CN, ß-CN, and a mix of α-LA and ß-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, ß-CN from small ruminant species (ovine and caprine) induced the highest levels of TNF-α, whereas PBMC incubated with αS2-CN from ovine milk and the mix of ß-LG and α-LA from all tested milk species had the lowest levels of TNF-α. Within the HL-EC group, production of IL-1ß was higher for bovine and ovine αS2-CN fractions and lower for caprine and ovine ß-CN and κ-CN. In the HL-EC group, IL-6 was higher in cultured PBMC incubated with αS2-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 ß-CN fraction in bovine milk. Catalase activity was higher in PBMC cultured with ß-CN isolated from bovine and caprine milk and with αS1-CN from ovine milk.

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 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-1ß, 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.

Quality of buffalo milk as affected by dietary protein level and flaxseed supplementation.

The aim of the present research was to evaluate the effects of protein level and flaxseed supplementation on the yield and quality of buffalo milk. In particular, the fatty acid profile of milk from buffalo cows subjected to different diets has been investigated. A 2×3 factorial design was tested with buffalo cows receiving 2 dietary crude protein (CP) and 3 flaxseed (FS) supplementation levels. Treatments were (1) low dietary CP level [12% of dry matter (DM)] and no flaxseed supplementation (LP); (2) low dietary CP level (12% of DM) and low flaxseed supplementation (500g/d) (LPFS500); (3) low dietary CP level (12% of DM) and moderate flaxseed supplementation (1,000g/d) (LPFS1000); (4) moderate dietary CP level (15% of DM) and no flaxseed supplementation (MP); (5) moderate dietary CP level (15% of DM) and low flaxseed supplementation (500g/d) (MPFS500); and (6) moderate dietary CP level (15% of DM) and moderate flaxseed supplementation (1,000g/d) (MPFS1000). Milk protein and casein were affected by flaxseed supplementation being higher in MP, intermediate in LP, and lower in flaxseed-supplemented diets. However, the results from the present study highlighted that low protein diets sustained milk yield, protein, and casein synthesis in milk when whole flaxseed was administered. Short-chain fatty acids, in particular C8:0 and C10:0, were the lowest in milk from buffalo cows fed the highest level of flaxseed supplementation. Medium-chain fatty acids were the lowest in FS1000, intermediate in FS500, and the highest in the HP and LP groups. Long-chain fatty acids were the highest in FS1000, intermediate in FS500 groups, and the lowest in milk from buffalo receiving no flaxseed supplementation. Protein level of the diet influenced the percentage of C18:0, which was higher in MP than LP groups. Total conjugated linoleic acid content evidenced the same trend of long-chain fatty acids, with an increase of about 7% in FL500 and of 22% in FL1000 than the control. Apart from protein level of the diet, atherogenic index, thrombogenic index, and n-6/n-3 were the lowest in FS1000 groups; thrombogenic index and n-6/n-3 were intermediate in milk from animals receiving FS500. Nutritional value of the acidic profile in buffalo milk is influenced by flaxseed supplementation, and its improvement reflects the level of dietary flaxseed supplementation.

Fatty acid profile of milk and Cacioricotta cheese from Italian Simmental cows as affected by dietary flaxseed supplementation.

The study aimed to determine the effects of adding flaxseed to the diet on the fatty acid profile of the milk of Italian Simmental cows and on the Cacioricotta cheese thereby produced. The experiment involved 24 Italian Simmental cows divided into 2 groups of 12 animals according to the diet fed: a control diet (CO) with no flaxseed supplementation, and a diet supplemented with whole flaxseed (FS). Milk yield and composition was not significantly changed by diet, whereas saturated fatty acids, monounsaturated fatty acids (MUFA), and polyunsaturated fatty acids (PUFA) were increased by flaxseed supplementation. Cows fed flaxseed showed higher percentages of long-chain fatty acids: in particular, linolenic acids, mainly represented by C18:3n-3, and n-3 series were higher in the FS group than in the CO group. The percentage of MUFA was higher by about 12% in FS than in CO, mainly due to the contribution of C18:1 cis-9. The percentage of conjugated linoleic acid (CLA) in milk was not significantly changed by flaxseed supplementation. Furthermore, atherogenic and thrombogenic indices were lower by about 30 and 16%, respectively, in the FS group compared with the CO group. The fatty acid profile of Cacioricotta cheese produced using Italian Simmental cow milk showed higher levels of MUFA, PUFA, and n-3, and improved atherogenic and thrombogenic indices in FS than in CO, confirming the ability to transfer beneficial molecules from milk into cheese. In particular, cheese-making technology contributed to the increased CLA content in Cacioricotta cheese.

Effect of stage of lactation on the immune competence of goat mammary gland.

The present research was undertaken to evaluate the effect of stage of lactation on immune competence of the goat mammary gland. The prevalence and etiology of intramammary infections and cytokine pattern were studied in individual caprine milk during lactation. A total of 1,200 caprine milk samples were collected during early, mid, and late lactation (less than 40 d, from 110 to 130 d, and more than 200 d in lactation, respectively) and analyzed for milk quality, bacterial cell load, and cytokine pattern. Contagious and environmental pathogens associated with intramammary infection were isolated from caprine milk. Nutritional components of caprine milk were affected by stage of lactation; fat, protein, casein, and whey protein increased along with lactation due to the lowest milk yield. Lactose content showed an opposite trend, with a reduced percentage passing from early to late lactation. The impaired rheological properties detected in early and late lactation milk were associated with a modification of milk quality. An impairment of microbial quality was detected at the end of lactation as a consequence of a major susceptibility of mammary gland to infections. The percentage of bacteriologically positive caprine milk samples was 44% throughout lactation. The incidence of bacteria associated with intramammary infection was different according to the stage of lactation with a major prevalence of Staphylococcus aureus, Enterobacteriaceae, Staphylococcus hyicus, and Streptococcus uberis in early lactation milk, Staphylococcus chromogenes, Streptococcus bovis, and Staphylococcus epidermidis in mid lactation, and Pseudomonas aeruginosa and Staphylococcus caprae in late lactation milk. The higher values of IL-1ß, tumor necrosis factor-α, and IL-6 were ascribed to the major prevalence of Staphylococcus aureus at the beginning of lactation, whereas a higher level of IL-10 was detected in mid-lactation caprine milk and correlated with Staphylococcus chromogenes. Monitoring the cytokine pattern in milk helps to understand the immune status of the mammary gland, its ability to cope with bacteria, and the evolution of infections.

Milk from different species: Relationship between protein fractions and inflammatory response in infants affected by generalized epilepsy.

The present study was undertaken to evaluate the effect of protein fractions from bovine, caprine, and ovine milk on production of cytokines and reactive oxygen species (ROS) and reactive nitrogen species (RNS) by cultured peripheral blood mononuclear cells (PMBC) from infants with generalized epilepsy. Bovine, caprine, and ovine bulk milks were pasteurized and analyzed for chemical composition. Then, PBMC were isolated from 10 patients with generalized epilepsy (5 males; mean age 33.6±5.4mo). Production of tumor necrosis factor-α (TNF-α), IL-10, IL-6, and IL-1ß was studied in cultured PBMC (from infants with epilepsy and controls) stimulated by bovine, caprine, and ovine milk and casein and whey protein fractions, and levels of ROS and RNS were measured in the culture supernatant. The ability of PBMC to secrete cytokines in response to milk and protein fraction stimulation may predict the secretion of soluble factor TNF-α in the bloodstream of challenged patients. Bovine, caprine, and ovine bulk milks induced low-level production of IL-10 by cultured PBMC in at least 50% of cases; the same behavior was observed in both casein and whey protein fractions for all species studied. Bovine and ovine milk and their casein fractions induced production of lower levels of IL-1ß in 80% of patients, whereas caprine milk and its casein fraction induced the highest levels in 80% of patients. The amount of IL-6 detected after stimulation of PBMC by milk and its fractions for all species was lower than that of other proinflammatory cytokines. In the bovine, total free radicals were higher in bulk milk and lower in the casein fraction, whereas the whey protein fraction showed an intermediate level; in caprine, ROS/RNS levels were not different among milk fractions, whereas ovine had higher levels for bulk milk and casein than the whey protein fraction. Lower levels of ROS/RNS detected in PBMC cultured with caprine milk fraction could be responsible for the lower levels of TNF-α cytokine in the corresponding fraction. Cytokines might be useful biomarkers to discriminate the effects of foods on the inflammatory response; dietary strategies could help in alleviating the negative effects of epilepsy in infants.

Identification of peptides in functional Scamorza ovine milk cheese.

Ovine bulk milk was used to produce Scamorza cheese with probiotics: either a mix of Bifidobacterium longum and Bifidobacterium lactis or Lactobacillus acidophilus as the probiotic strains. Peptides obtained from reverse phase-HPLC water-soluble extract of Scamorza cheeses were analyzed using a quadrupole time-of-flight liquid chromatography-mass spectrometry system. Identified fragments were derived from casein hydrolysis or probiotic bacterial enzymes; some of the fragments showed encrypted peptide sequences that shared structural homology with previously described bioactive peptides in ovine milk and dairy products. Bifidobacterium longum and B. lactis showed greater proteolytic potential both in terms of level of pH 4.6 water-soluble nitrogen extract and ability to generate peptides with potential biofunctionality. Fragments deriving from microbial enzymes may be regarded as tracing fragments useful for monitoring probiotic activity in functional Scamorza cheese.

Activities of indigenous proteolytic enzymes in caprine milk of different somatic cell counts.

Individual caprine milk with different somatic cell counts (SCC) were studied with the aim of investigating the percentage distribution of leukocyte cell types and the activities of indigenous proteolytic enzymes; proteolysis of casein was also studied in relation to cell type following recovery from milk. The experiment was conducted on 5 intensively managed dairy flocks of Garganica goats; on the basis of SCC, the experimental groups were denoted low (L-SCC; <700,000 cells/mL), medium (M-SCC; from 701,000 to 1,500,000 cells/mL), and high (H-SCC; >1,501,000 cells/mL) SCC. Leukocyte distribution differed between groups; polymorphonuclear neutrophilic leukocytes were higher in M-SCC and H-SCC milk samples, the percentage macrophages was the highest in H-SCC, and levels of nonviable cells significantly decreased with increasing SCC. Activities of all the main proteolytic enzymes were affected by SCC; plasmin activity was the highest in H-SCC milk and the lowest in L-SCC, and elastase and cathepsin D activities were the highest in M-SCC. Somatic cell count influenced casein hydrolysis patterns, with less intact α- and ß-casein in H-SCC milk. Higher levels of low electrophoretic mobility peptides were detected in sodium caseinate incubated with leukocytes isolated from L-SCC milk, independent of cell type, whereas among cells recovered from M-SCC milk, macrophages yielded the highest levels of low electrophoretic mobility peptides from sodium caseinate. The level of high electrophoretic mobility peptides was higher in sodium caseinate incubated with polymorphonuclear neutrophilic leukocytes and macrophages isolated from M-SCC, whereas the same fraction of peptides was always the highest, independent of leukocyte type, for cells recovered from H-SCC milk. In caprine milk, a level of 700,000 cells/mL represented the threshold for changes in leukocyte distribution, which is presumably related to the immune status of the mammary gland. Differences in the profile of indigenous lysosomal proteolytic enzymes in caprine milk may influence the integrity of casein based on proteolysis patterns of sodium caseinate incubated with isolated and lysed leukocyte types.

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.

Composition and sensory profiling of probiotic Scamorza ewe milk cheese.

The present study aimed to assess the effect of the addition of different usually recognized as probiotic bacterial strains on chemical composition and sensory properties of Scamorza cheese manufactured from ewe milk. To define the sensory profile of Scamorza cheese, a qualitative and quantitative reference frame specific for a pasta filata cheese was constructed. According to the presence of probiotic bacteria, cheeses were denoted S-BB for Scamorza cheese made using a mix of Bifidobacterium longum 46 and Bifidobacterium lactis BB-12, and S-LA for Scamorza cheese made using Lactobacillus acidophilus LA-5. The designation for control Scamorza cheese was S-CO. Analyses were performed at 15d of ripening. The moisture content of Scamorza ewe milk cheese ranged between 44.61 and 47.16% (wt/wt), showing higher values in S-CO and S-BB cheeses than in S-LA cheese; the fat percentage ranged between 25.43 and 28.68% (wt/wt), showing higher value in S-LA cheese. The NaCl percentage in Scamorza cheese from ewe milk was 1.75 ± 0.04% (wt/wt). Protein and casein percentages were the highest in Scamorza cheese containing a mix of bifidobacteria; also, the percentage of the proteose-peptone fraction showed the highest value in S-BB, highlighting the major proteolysis carried out by enzymes associated with B. longum and B. lactis strains. Texture and appearance attributes were able to differentiate probiotic bacteria-added cheeses from the untreated control product. In particular, S-BB and S-LA Scamorza cheeses showed higher color uniformity compared with S-CO cheese. Furthermore, the control cheese showed higher yellowness and lower structure uniformity than S-BB. The control product was less creamy and grainy than S-BB; conversely, the inclusion of probiotics into the cheese determined lower adhesivity and friability in S-BB and S-LA than in S-CO. This study allowed the definition of the principal composition and sensory properties of Scamorza ewe milk cheese. The specific quantitative vocabulary for sensory analysis and reference frame for assessor training also established in this study should be implemented to systematically monitor the quality of this new typology of ewe milk cheese.

Physicochemical properties of Scamorza ewe milk cheese manufactured with different probiotic cultures.

The present study was undertaken to produce functional Scamorza cheese from Gentile di Puglia ewe milk by incorporating probiotic strains into the cheese matrix and to evaluate the physicochemical characteristics of Scamorza ewe milk cheese. Gentile di Puglia ewe bulk milk was used for Scamorza cheese production. Cheeses were denoted S-CO for control Scamorza cheese, S-BB for Scamorza cheese made using a mix of Bifidobacterium longum and Bifidobacterium lactis, and S-LA for Scamorza cheese made using Lactobacillus acidophilus as probiotic strain. Cheeses were analyzed at 1, 7, and 15 d of ripening. Probiotic cell recovery in cheese was 7.55 ± 0.07 log10 cfu/g and 9.09 ± 0.04 log10 cfu/g in S-LA and S-BB cheese, respectively; probiotic cheeses also displayed the highest levels of lactic microflora. Reverse-phase HPLC chromatograms of the water-soluble nitrogen fraction showed a more complex profile in S-BB, with distinctive peaks in the early-eluting zone. The matured Scamorza cheese containing the mix of B. longum and B. lactis was characterized by significantly higher levels of Gln, Ser, Arg, Ile, and Leu, whereas cheese containing Lb. acidophilus was characterized by higher levels of Tyr and Met. Total FFA content was the highest in S-LA, intermediate in S-BB, and the lowest in S-CO cheese; in particular, Scamorza cheese containing Lb. acidophilus showed the highest level of vaccenic acid, oleic acid, and total conjugated linoleic acid. Probiotic bacteria survived through the technological phases of pasta filata cheese production, maintained their specific metabolic pathways, and conferred functional properties to Scamorza ewe milk cheese.

Dietary glutamine enhances immune responses of dairy cows under high ambient temperature.

The objective of the study was to determine the effects of adding Gln or Gln in combination with flaxseed to the diet on milk production and immune response of dairy cows during the summer season. The experiment lasted 60 d and involved 24 lactating Friesian cows, divided into 4 groups of 6 animals, fed as follows: (1) control diet, (2) control diet supplemented with 160 g/d of rumen-protected Gln (GLN), (3) control diet supplemented with whole flaxseed (FS), and (4) control diet supplemented with both flaxseed and Gln (FS+GLN). Individual milk samples were collected at morning and afternoon milkings every 14 d throughout the experiment and analyzed for pH, total protein, casein, fat, lactose content, and somatic cell count. Milk samples were also analyzed for milk fatty acid profile. Monitoring of cow immune responses was carried out by the determination of lymphocyte response to phytohemagglutinin in vivo on each animal by measurement of skin-fold thickness at site of phytohemagglutinin injection. A humoral response to chicken egg albumin (OVA) was determined following a subcutaneous injection with OVA. Plasma samples were measured for the presence of anti-OVA IgG, IL-1ß, IL-6, and IL-10. Results indicate that Gln administration can be used to support dairy cows under a hot climate, because Gln was able to enhance cow immune reactions in terms of a strengthening of cell-mediated immune response, which is weakened in cows under heat stress. In addition, an increase in milk and fat, protein, and casein yields in response to GLN was observed. When administrating Gln in combination with flaxseed, milk production and milk fat profile did not further increase compared with FS milk, and a reduction in immune reactivity compared with FS and GLN cows was found.

Effects of extended transition milk feeding on blood metabolites of female Holstein dairy calves at 3 weeks of age: a liquid chromatography with tandem mass spectrometry-based metabolomics approach.

Transition milk (TRM) is a rich source of bioactive components that promotes intestinal development and growth, and reduces the susceptibility to diarrhoea in calves. The objective of this study was to characterise the effects of replacing pasteurised waste milk (none-saleable milk containing antibiotic and/or drug residues) with pasteurised TRM for 3 wk on blood metabolites of dairy calves at 21 d of age. A total of 84 healthy newborn female Holstein calves was blocked by birth order and assigned randomly to four treatment groups with partial replacement of pasteurised waste milk by TRM (second milking after parturition) at 0 (0 L/day TRM + 6 L/day milk), 0.5 (0.5 L/day TRM + 5.5 L/day milk), 1 (1 L/day TRM + 5 L/day milk), or 2 L (2 L/day TRM + 4 L/day milk) for a 21-day period. Serum metabolome was determined by liquid chromatography with tandem mass spectrometry-based metabolomics analysis on a subset of 26 randomly selected individuals from calves fed pasteurised waste milk (CON, 6 L/d milk; n = 13) or TRM (2 L/d TRM + 4 L/d milk; n = 13) at 21 d of age. The identified metabolites (194 out of 265) were categorised according to chemical class and the number of metabolites per class in the serum, amongst which glycerophospholipids 16% (n = 43), fatty acyls 7% (n = 19), organic acids 7% (n = 18), organic heterocyclic compounds 5% (n = 13), benzenoids 5% (n = 12), sphingolipids 5% (n = 12), organic oxygen compounds 4% (n = 11), and nucleic acids 3% (n = 9), were the predominant types. Significant differences in metabolites were determined by the volcano plot. Applying the volcano plot, only two metabolites (ceramide and phosphatidylserine) were significantly different between CON and TRM. Overall, our results suggested that prolonged TRM feeding for 3 wk had little effect on the serum metabolome of the dairy calves. We speculate that the potential effects of feeding TRM for 3 wk compared with waste milk were spatially limited to affect the composition of the local gut microbial community and the growth or function of the intestinal epithelium, not allowing detection of the likely effects in the serum through a metabolomic approach.

Immune competence of the mammary gland as affected by somatic cell and pathogenic bacteria in ewes with subclinical mastitis.

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 <300,000 cells/mL, from 300,000 to 500,000 cells/mL, from 501,000 to 1,000,000 cells/mL, from 1,001,000 to 2,000,000 cells/mL, and >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 <300,000 cells/mL; starting from 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.