Technical note: immunomagnetic procedure for positive selection of macrophages in ovine milk.

A simple immunomagnetic procedure was developed to select macrophages from ovine milk by using a non-specific magnetic positive separation technique. Samples of ewe bulk milk were collected during early, mid, and late lactation; milk samples were centrifuged at 2,000 x g for 30 min at 4 degrees C; the fatty fraction and supernatant were removed, and each pellet was dissolved in 500 microL of pH 7.4 phosphate buffered saline + 0.02% NaN(3). Cells were targeted for selection by using mouse-IgG anti-ovine macrophages. Several trials, testing 2 different fluorochrome-conjugated antibodies [i.e., mouse anti-human CD14:R-Phycoerythrin (RPE) (MCA1568PE, Serotec) and F(ab')2 rabbit anti-mouse IgG:RPE (STAR12A, Serotec)] and 3 different labeling procedures, were performed to evaluate the purity of samples by flow cytometry. A morphological test was carried out by direct microscopic count in enriched fraction smears stained with May-Grünwald-Giemsa stain to confirm the presence of macrophages. The method described in the present technical note can be considered an innovative application to obtain a single-cell population of high purity selected from all the somatic cells in milk

Changes occurring in immune responsiveness of single- and twin-bearing Comisana ewes during the transition period.

Changes induced by twin and single lambing in the immune response of 16 periparturient Comisana ewes were studied. Cell-mediated immune responses were evaluated by means of skin tests performed from 3 wk before and up to d 35 after parturition. At d 21 and 7 before lambing, the sheep received an intramuscular injection of the antigen keyhole limpet hemocyanin (KLH), to which the animals had not been previously exposed, to determine their humoral immune response. Starting 3 wk before lambing and up to d 35 postlambing, the ewes were sampled to determine the plasma concentrations of anti-KLH antibody (IgG), IL-6, and IL-1 beta. From parturition through d 35 postpartum, individual milk samples were collected for determination of anti-KLH IgG titers and IL-6 and IL-1beta concentrations by means of a capture ELISA. The number of lambs born affected IL-6 concentrations in ewe plasma; IL-6 secretion always was higher in ewes birthing twins than in single-lambing ewes. Apart from the number of lambs born, the concentrations of plasma IL-6 in ewes were higher at lambing than at d 21 antepartum and at d 35 postpartum. An interaction of number of lambs born x time of sampling was observed for plasma antibody titers to KLH. The IgG concentrations were significantly higher in single-bearing ewes than in twin-bearing ewes before parturition and were very similar across groups after parturition. A time effect was found for the cell-mediated immune response and for anti-KLH IgG concentrations in milk, such that at parturition, cellular responses were lowest, and the anti-KLH IgG concentration was highest. A significant correlation was found for IgG titers to KLH in plasma and milk. Results indicate that IL-6 concentrations in blood can be considered a reliable indicator of stress connected to lambing and that the mammary gland is a microenvironment unrelated to blood stream with respect to interleukins expression. In contrast, a relationship was found for the IgG secretions in milk and blood, which suggests that the assessment of humoral immune status may be combined with milking routine in dairy animals

Influence of ventilation regimen on micro-environment and on ewe welfare and milk yield in summer

The effects of ventilation regimen on air quality, and on the welfare and production performance of thirty-six Comisanaewes were assessed in a 6-week trial conducted during the summer of 2002. Animals were divided into three groups of12, and subjected to the following treatments: low ventilation regimen providing a mean ventilation rate (VR) of 35 m3/hper ewe, split in 30 min ventilation cycles at an air speed of 2 m/s (LOV-30); moderate ventilation regimen (VR = 70m3/h per ewe) split in 30 min ventilation cycles at an air speed of 4 m/s (MOV-30); moderate ventilation regimen (VR =70 m3/h per ewe) split in 60 min ventilation cycles at an air speed of 2 m/s (MOV-60). Air concentrations of microorganisms,dust, and gaseous pollutants were measured twice weekly. Respiration rate (RR) and rectal temperature (RT)were monitored throughout the trial at 0830 and at 1400. Behavioral traits of ewes were recorded twice per week from0900 to 1200 and from 1500 to 1800. Cell-mediated immune response to phytohemagglutinin (PHA) and humoralimmune response to chicken egg albumin were determined. At d 37 ewes were injected with porcine ACTH, and subjectedto blood sampling for evaluation of cortisol concentrations immediately before and 1, 2 and 4 h after ACTH injection.Milk yield was recorded daily. Individual milk samples were analyzed for composition, renneting parameters, somaticcell count (SCC), and bacteriological characteristics. Averages of maximum THI were about 3 points higher in the LOV-30 and the MOV-30 than in the MOV-60 room, whereas no differences emerged in the air concentrations of dust, gaseouspollutants and microorganisms. Significant interactions of treatment x time (P < 0.05) were found for respiration rate,and for the time the ewes spent lying, idling and eating in the afternoon during weeks 2 and 3 of the study period.Significant effects of ventilation regimen x time (P < 0.05) were also observed for milk yield and milk renneting parameters,the LOV-30 ewes giving smaller volumes of milk with a deteriorated coagulating behavior than those of the MOV-60 group during the second half of the trial. No significant differences emerged in ewe immune and endocrine responses.Results show that ventilation regimen had a moderate impact on ewe behavior, physiology and production performance.This experiment suggests that the length of ventilation cycles and air speed, together with ventilation rate, arecritical for efficient ventilation regimens

Contribution of macrophages to proteolysis and plasmin activity in ewe bulk milk.

A total of 225 bulk sheep milk samples were collected from 5 intensively managed flocks during early, mid, and late lactation to assess the contribution of macrophages to the regulation of the plasmin-plasminogen system. Samples were analyzed for composition, somatic cell counts, milk renneting characteristics, and for plasmin (PL), plasminogen (PG), and plasminogen activators (PA) activities. Isolation of macrophages from milk was performed using a magnetic positive separation and mouse antiovine macrophage antibody; separated cells were lysed by several freeze-thaw cycles, and activity of urokinase PA (u-PA) was determined. Plasmin activity decreased during lactation (42.06 +/- 0.66, early; 31.29 +/- 0.66, mid; 28.19 +/- 0.66 U/mL, late). The reduction in PL activity recorded in the mid and late lactation milk matched the increase in PG:PL ratio. The activity of PA increased throughout lactation; the highest value being recorded in the late lactation milk (260.20 +/- 8.66 U/mL). Counts of isolated and concentrated macrophages were higher in early and mid lactation milk (3.89 +/- 0.08 and 3.98 +/- 0.08 log10 cells/mL, respectively) than in late lactation milk (3.42 +/- 0.08 log10 cells/mL). Stage of lactation did not influence the activity of u-PA detected in isolated macrophages. The activity of u-PA associated with isolated milk macrophages only minimally contributed to total PA activity detected in milk. Proteolytic enzymes, associated with isolated macrophages, act on alpha-casein hydrolysis, as shown by urea-PAGE electrophoresis analysis. Somatic cell counts did not exceed 600,000 cells/mL, and this threshold can be considered a good index of health status of the flock and of the ability of milk to being processed. Our results lend support to the hypothesis that macrophages in ewe bulk milk from healthy flocks only slightly contribute to the activation of the PL-PG system

Relationship between cortisol response to stress and behavior, immune profile, and production performance of dairy ewes.

The existence of a relationship between cortisol levels, after an acute stress, and behavioral activities, immunological profile, and production performance in sheep was studied. An initial flock of 30 Comisana ewes was involved in the experiment, and each of the 30 ewes was individually subjected to an isolation test in a novel environment. Subsequently, from the initial flock, 2 groups of 8 Comisana ewes were each retrospectively selected, and the animals were divided, according to their cortisol concentration 10 min after the isolation test, into high cortisol (HC) ewes, having a peak of cortisol concentration >90 ng/mL (average: 119.3 ng/mL +/- 11.8), and low cortisol (LC) ewes having a peak of cortisol concentration <80 ng/mL (average: 52.4+/-11.8). During the isolation test, the behavior of each animal was video-recorded and behavioral activities were registered. Blood samples were collected before the isolation test, immediately after the test (10 min), and at 60, 120, 300 min, 24 h, and 48 h after the test to evaluate percentages of T-helper (CD4(+)) and T-cytotoxic (CD8(+)) cells, CD4(+)/CD8(+) ratio, and IL-1beta and IL-6 levels. The ewes were milked for 3 d after the isolation test to determine cortisol levels and IL-1beta and IL-6 concentrations in whey. Milk yield was recorded at each milking, and milk samples were analyzed for pH, nutritional parameters, renneting properties, and somatic cell count. During the isolation test, HC ewes exhibited a shorter duration of movement and fewer bleats than LC ewes. The average plasma IL-1beta concentration was higher in HC than in LC ewes. The average whey IL-1beta and IL-6 concentrations were higher in whey from HC ewes than in LC ewes. A positive correlation emerged between plasma and whey IL-1beta concentrations. The average CD4(+)/CD8(+) ratio in blood was lower in HC than in LC ewes. Time from isolation affected the CD4(+)/CD8(+) ratio: at 120 min, the CD4(+)/CD8(+) ratio increased compared with that at 10 min after isolation and then decreased until 300 min after isolation. On average, ewes with low cortisol concentrations showed higher milk production and lower SCC than ewes with high cortisol concentrations. Results suggest that plasma cortisol concentration is connected to the behavioral response and immune competence of dairy ewes and cytokine concentrations. Both whey IL-1beta and IL-6 can be considered reliable indicators of the magnitude of hypothalamic-pituitary-adrenal axis activation. The stress-induced changes in CD4(+)/CD8(+) ratio are critical for controlling disease incidence and planning appropriate vaccination programs. High reactivity of the hypothalamic-pituitary-adrenal axis is also associated with a reduction in milk production and an increased predisposition to develop intramammary inflammatory processes

Technical note: Rapid method for determination of amino acids in milk.

A rapid method for measurement of amino acids in milk was developed and validated. The method included a first step of milk protein hydrolysis, followed by the derivatization and separation of amino acids by HPLC. Six combinations of hydrolysis agent and temperature-time conditions were compared with a reference method; derivatization procedures as well as HPLC separation were improved. Hydrolysis of milk samples with 6 N HCl at 160 degrees C for 60 min resulted in no significantly differences compared with the reference method but allowed the analysis of a greater number of milk samples in a short time. In addition, this method was characterized by high precision, low repeatability uncertainty, and high accuracy for all amino acids evaluated; the recovery mean value of the single amino acids was 98.38%. The proposed method is, therefore, accurate, simple, rapid, and suitable for large numbers of milk samples

Stress-induced changes in immune response of dairy ewes

two groups of 16 Comisana ewes were selected from a group of 30, and divided, according to their cortisol secretion after isolation in a novel environment, into HC ewes, having a cortisol secretion >90 ng/mL, and LC ewes having a cortisol secretion <80 ng/mL. Blood samples were collected immediately before and immediately after isolation, and 60, 120, 300min and then 24h and 48h after isolation, to evaluate the percentage of lymphocyte T-helper (CD4+) and lymphocyte T-cytotoxic (CD8+) by flow cytometry analysis, and IL-1β and IL-6 concentrations by ELISA. The day before the isolation test, the ewes were milked in the afternoon at 1500h (-19h from isolation), and then milked for the three days starting from the day of the isolation test (at 0, 5, 24, 29, 48 and 53h from isolation) to evaluate milk cortisol secretion, and IL-1β and IL-6 concentrations. Results suggest that the levels of cortisol secretions can influence the immune competence of dairy ewes and cytokines concentrations. Milk cytokine concentrations, and not milk cortisol concentrations, can be considered indicators of the magnitude of the hypothalamic-pituitary-adrenal axis activation

Monitoring the on-farm welfare of sheep and goats

Schemes for on-farm welfare assessment in sheep and goats are not yet available. Factors responsible for this lack of availability are discussed in the first part of this review. Requisites for reliable methods of welfare assessment to be used in sheep and goat farms are discussed, taking into account the peculiarities of the small ruminant production systems in terms of flock management and farm location. Some housing parameters related to structures, design and micro-environment are reviewed and could be included in schemes of welfare assessment on farm. Human-animal interaction, health status of the animals in terms of body condition scoring, skin and hair conditions, lameness and injuries, and management practices in relation to cleanliness, animal handling and moving, milking procedures and abnormal behaviours, are proposed as potential animal-based indicators. When available, validity, reliability and feasibility of the proposed parameters are discussed. The voluntary adhesion to welfare standards in Sardinia and the application of a scientifically validated protocol in Basilicata are reported as examples of practical experiences

Consumer acceptance and sensory evaluation of Monti Dauni Meridionali Caciocavallo cheese

Twelve Caciocavallo cheeses were collected from 6 factories (A, B, C, D, E, F) located in the Monti Dauni Meridionali area (Southern Italy) that adopted different protocols for cheese production. A total of 160 consumers were involved in the sensory evaluation of Caciocavallo cheese after 180 d of ripening. Cheese attributes were used to describe the flavor, texture, and appearance of cheeses. The highest scores for the shiny attribute were assigned to cheeses B, C, and E, whereas color intensity was the highest in cheeses B, D, and F. Strength, salty, and piquant attributes were higher in cheeses F and A because of the use of raw milk (F), rennet paste (A), and percentage of salt in the brine (A, F). Consumers perceived a more granular structure during the second half of chewing of Caciocavallo cheese F, as evidenced by the highest value for the grainy attribute. A positive correlation was found between overall flavor and odor intensity and water-soluble nitrogen, low molecular weight peptides, and free fatty acids and between piquant and butyric and caproic acids. A principal components analysis applied to the sensory attributes accounted for 65% of the total variance. The score plot showed that cheeses F and A were located in a well-defined zone of the plot, with cheeses in this zone displaying higher levels of strength, piquant, and salty attributes. The preference test assigned 40% of the preference to Caciocavallo cheese A, 38% to cheese F, 9% to cheese E, 8% to cheese D, and 7% to cheeses B and C. Sensory evaluation of Monti Dauni Meridionali Caciocavallo cheeses is a useful analysis to highlight the principal attributes able to influence consumers' liking that are related to biochemical features of the cheese

Effect of solar radiation and flaxseed supplementation on milk production and fatty acid profile of lactating ewes under high ambient temperature

Abstract The objectives of this study were to evaluate the effects of protection from solar radiation and whole flaxseed supplementation on milk yield and milk fatty acid profile in lactating ewes exposed to high ambient temperature. The experiment was conducted during summer and involved 40 ewes divided into 4 groups. The ewes were either exposed (not offered shade) or protected from solar radiation (offered shade). For each solar radiation treatment, ewes were supplemented with whole flaxseed or not. Milk samples from each ewe were collected at the morning and afternoon milking every week, and analyzed for pH, total protein, casein, fat, and lactose content, somatic cell count, and renneting parameters (clotting time, rate of clot formation, and clot firmness after 30min). At the beginning of the experiment, and then at d 23 and 44, milk samples were analyzed for milk fatty acids using gas chromatography. Flaxseed supplementation significantly increased milk yield, fat, protein, and casein yields, and somatic cell count, and increased fat and lactose contents of milk. A decrease of saturated fatty acids from C6:0 to C16:0 and an increase of C18:1 trans -11 and C18:2 cis- 9, trans -11 was observed in milk from flaxseed-supplemented ewes. Flaxseed supplementation decreased saturated fatty acids content and increased total monounsaturated fatty acids content, the total content of isomers of conjugated linoleic acid, and polyunsaturated fatty acids content in milk. Flaxseed also increased the α-linolenic acid content of milk. As a result, milk from supplemented groups showed an increase in n-3 fatty acid content. Flaxseed supplementation decreased short-chain and medium-chain fatty acids, and increased long-chain fatty acid content of milk. On average, flaxseed supplementation increased the C18:2 cis -9, trans -11/C18:1 trans -11 Δ 9 -desaturase index starting from d 23 of the experiment, in correspondence with the highest C18:2 cis -9, trans -11 content of milk from flaxseed-supplemented ewes. Flaxseed decreased atherogenic and thrombogenic indices of milk. Protection from solar radiation during summer did not improve yield and composition of ewe milk. Nevertheless, milk from ewes exposed to solar radiation showed decreased long-chain fatty acid and polyunsaturated fatty acids contents, and in particular, decreased vaccenic acid, rumenic acid, and total conjugated linoleic acid contents