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The aim of the present study was to determine the role of milk endogenous proteolytic enzymes in sheep milk cheesemaking ability during lactation. Plasmin, plasminogen, and plasminogen activator in ewe bulk milk were not significantly affected by stage of lactation, probably because of the good health of the ewe udders throughout lactation as indicated by somatic cell count, which never exceeded 600,000 cells/mL. Elastase content increased significantly during lactation, whereas cathepsin showed the greatest content in mid lactation. Early and mid lactation milk showed impaired renneting parameter compared with late lactation milk, probably because of greater alpha-casein degradation, brought about by cathepsin, and lesser fat and casein (CN) milk contents. Changes in macrophage and neutrophil levels in ewe bulk milk during lactation were also investigated. Macrophages minimally contributed to leukocyte cell count in milk and had the greatest levels at the beginning of lactation. An opposite trend was recorded for polymorphonuclear neutrophilic leucocytes (PMNL) that increased throughout lactation, showing the greatest value in late lactation. Urea-PAGE of sodium caseinate (NaCN) incubated with isolated and concentrated PMNL at 37 degrees C after 48 h at pH 8 showed massive casein degradation that could be ascribed to proteases yielded by PMNL. The increase of PMNL percentage and elastase content in milk, despite the relatively low SCC, suggests that PMNL and elastase underwent a physiological increase associated to the remodeling of mammary gland in late lactation.

Effects of ventilation rate and of dietary protein level in an intensive dairy sheep system on the features of Canestrato Pugliese cheese were studied. Cheeses were manufactured from the bulk milk obtained from ewes subjected to four different experimental treatments: (1) low dietary crude protein (CP) of 13% in dry matter (DM) at a low ventilation rate (23.5 m3/h per ewe) (LPLV); (2) low dietary CP at a moderate ventilation rate (47 m3/h per ewe) (LPMV), (3) moderate dietary CP of 16% DM at a low ventilation rate (MPLV); and (4) moderate dietary CP dietary at a moderate ventilation rate (MPMV). Bulk milk and cheeses (at 1, 15, 45 and 90 d of ripening) were analysed for chemical composition, N fractions and plasmin-plasminogen activities. The pH 4.6-soluble and insoluble N fractions were analysed by urea-PAGE. Bulk milk from ewes receiving the low CP diet displayed higher casein and lower urea contents. Ewes subjected to the low ventilation rate displayed a higher plasminogen activity in milk, whereas no differences emerged among treatments in the conversion of zymogen to plasmin. During ripening the plasmin-plasminogen system in cheese did not display significant changes across treatments. At 90 d of ripening, the cheese produced with milk from ewes receiving the low CP diet and exposed to the low ventilation rate displayed more markedly stained bands in the area of gamma-caseins in pH 4.6-insoluble N fraction. As from 15 d of ripening, the pH 4.6-soluble fraction showed a greater number of bands in the LPMV and MPMV than in the LPLV and MPLV cheeses. The results showed that Canestrato Pugliese cheese manufactured with milk from ewes fed the low CP diet and exposed to the moderate ventilation rate was characterized by higher protein and casein content in the fresh cheese and by a greater proteolysis after 90 d of ripening.

The experiment, which lasted 53 d, was conducted during the winter (February and March) of 2004 and used 48 Comisana ewes in mid lactation. A 2 x 2 factorial design was used, with ewes receiving two levels of dietary crude protein (CP) (moderate, 16% CP v. low, 13% CP) in the dry matter (DM) and being exposed to two ventilation rates (moderate, 47 m3/h v. low, 23.5 m3/h per ewe) for each dietary treatment. Air concentrations of NH3 and of microorganisms were measured twice weekly. Milk yield was recorded daily. Individual milk samples were analysed weekly for composition and fortnightly for bacteriological characteristics. After the last milk sampling (day 49 of the study period), four animals from each group were placed in a metabolism box and their individual faeces and urine were collected for three consecutive days. Amounts of urine and faeces excreted, and urinary and faecal N outputs were measured. The 16% CP diet resulted in a lower milk casein content and a higher milk urea concentration than the 13% CP diet, as well as in a reduced gross efficiency of utilization of dietary N, a greater amount of N excreted and a higher total coliform concentration in milk. The moderate ventilation rate resulted in higher yields of milk, irrespective of CP content. Significant interactions of CP level x ventilation rate were found for the amounts of urine, of total water and of faecal N, and for mesophilic concentration in milk, the highest values being displayed by the ewes fed the 16% CP diet and exposed to the low ventilation rate. The moderate dietary CP level and low ventilation rate had a deleterious effect on air concentrations of microorganisms and ammonia. Results suggested that a reduction of dietary CP level from 16 to 13% of DM had no detrimental effect on ewe milk yield in mid lactation and could even improve some of its nutritional and hygienic characteristics. Our findings also indicate that the choice of a proper ventilation rate is critical for high efficiency of production in the lactating ewe, especially in intensively managed flocks receiving diets high in CP.

The relationships between welfare and udder health indicators were investigated in 120 midlactation Comisana ewes over a 90-day period. The welfare indicators monitored were the humoral response to chicken egg albumin (OVA), the cell-mediated immune response to phytohemagglutinin, and the plasma cortisol levels. Changes in milk components, in somatic cell count (SCC) and in bacterial count, in leukocyte populations and in anti-OVA IgG concentration were used to monitor the ewe udder health. Correlations between welfare and udder health indicators were then tested. The skinfold thickness, representative of the cell-mediated immune response, was correlated positively with the percentage of neutrophils in milk and negatively with the chloride content, the percentage of lymphocytes and anti-OVA IgG concentrations in milk. The plasma antibody titres to OVA, representative of the humoral immune response, correlated positively with the anti-OVA IgG levels in milk and negatively with the lactose content and to the percentage of neutrophils. Plasma cortisol levels correlated positively with the chloride content in milk. Our study demonstrates that information about the immune condition of animals can be successfully achieved by monitoring the immunological responses of the mammary gland. The detection of reliable indicators of animal welfare in milk can help in reducing the stress from animal handling and blood sampling from lactating animals.

The influence of three different ventilation regimens on air pollution in sheep houses and on the quality of ewe milk and of Canestrato Pugliese cheese was investigated during the summer season. The experimental treatments were low ventilation regimen (VR=35 m3/h per ewe) split in 30-min ventilation cycles (LOV-30); moderate ventilation regimen (VR=70 m3/h per ewe) split in 30-min ventilation cycles (MOV-30); moderate ventilation regimen (VR=70 m3/h per ewe) split in 60-min ventilation cycles (MOV-60). The LOV-30 milk had higher microbial load and bulk milk somatic cell count (BMSCC) and resulted in a weaker casein matrix in the curd compared with the MOV-30 and MOV-60 treatments. At 45 d of ripening, the LOV-30 cheeses had a lower casein content and higher non-casein nitrogen (NCN) and water-soluble nitrogen (WSN) contents than the MOV-30 and MOV-60 cheeses. Urea-polyacrylamide gel electrophoresis (urea-PAGE) of the pH 4.6-soluble N extract showed that the MOV-60 cheeses had fewer bands derived from casein (CN) hydrolysis than the LOV-30 or MOV-30 cheeses, despite its having exhibited the highest plasmin (PL) activity levels. Our results suggest that the ventilation regimen is critical in dairy sheep housing for optimizing the hygienic quality of ewe milk and the proteolytic processes occurring in Canestrato Pugliese cheese during ripening.

A total of 120 milk samples were collected from Comisana ewes throughout lactation. The ewes were ranked into two somatic cell count (SCC) categories: normal milk (N Milk) with SCC lower than 5.00x 10(5)/ml and high somatic cell milk (HSC Milk) with SCC higher than 1.00 x 10(6)/ml. Milk samples were analysed in triplicate for pH, fat and protein contents, renneting parameters, and plasmin and plasminogen activities. The peptide profile due to total proteolytic activity (endogenous and exogenous enzymes) on alpha- and beta-CNs were determined using urea-PAGE on sodium caseinate (pH 8.0 and pH 5.0) incubated at 37 degrees C for 4 d after sampling. The peptide profile due to non-plasmin enzyme activities at pH 5.0 was also determined using urea-PAGE. Plasmin activity was higher in the HSC milk than in the N milk throughout the study period. A decrease in plasmin activity was observed in the N milk during mid-lactation, which was probably related to decrease in pH, and in the HSC milk during late lactation, which may be ascribed to an enhanced influx of plasmin inhibitors from the blood stream. Proteolytic patterns in Comisana ewe milk were mainly affected by plasmin activity that increased with the SCC in milk. Also non-plasmin proteolytic activity was strongly enhanced by elevated SCC and resulted in a higher degradation of alpha-casein than of beta-casein. In general, plasmin activity did not increase with the advancement of lactation and exhibited a different trend in HSC and N milk, suggesting that physiological factors did not play a key role in regulating the plasminogen-plasmin system in ewes' milk. Plasmin activity, detected with the colorimetric assay was consistent with proteolytic activity on sodium caseinate shown in urea-PAGE electrophoregram.

Effects of ventilation regimen on the quality of ewes' milk and on proteolysis in Canestrato Pugliese cheese during ripening were studied. Cheeses were manufactured from the bulk milk of Comisana ewes subjected to three different ventilation regimens, which were designated low (LOV, 23 m3/h per ewe), moderate (MOV, 47 m3/h per ewe) and programmed ventilation regimen (PROV, 73 m3/h per ewe; fan set to maintain 70% relative humidity). Bulk milk was analysed for chemical and microbial composition, renneting parameters and plasmin-plasminogen activities. At 1, 15, 30 and 45 d of ripening, the cheeses were analysed for gross chemical composition, nitrogen fractions, and plasmin and plasminogen activities. The pH 4.6-insoluble nitrogen fractions were analysed by urea-PAGE. Free amino acid content was determined at the end of ripening. Lower concentrations of bulk milk somatic cell count (BMSCC) and of mesophilic bacteria were found in the MOV group than in the LOV and the PROV groups. A lower plasminogen (PG) to plasmin (PL) ratio (PG/PL) was observed in the MOV and PROV than in the LOV cheeses. Irrespective of treatment, PL activity in cheeses was higher at 15d of ripening, while a sudden decrease of PL and PG activities was observed at 30 d, which was associated with a marked increase in non-protein nitrogen. The peptide profile characterized in the urea-PAGE showed a greater intensity of alpha- and beta-CN hydrolysis in the MOV than in the PROV and LOV cheeses. The results provide evidence that a proper ventilation regimen is critical for optimizing the hygienic quality of milk and the proteolysis of Canestrato Pugliese cheese during ripening.

A 6-wk trial was performed with thirty-six lactating Comisana ewes during the summer of 2001. The animals were divided into three groups of 12, which were designated low (LVR), moderate (MVR), and programmed (PROGR) ventilation regimens. In LVR and MVR rooms, fans provided 10 ventilation cycles of 12.5 and 25 min/h, respectively, whereas in the PROGR room, the fan was programmed to operate at 30 degrees C air temperature and 70% relative humidity. Mean ventilation rates were 33, 66, and 173 m3/h per ewe in LVR, MVR, and PROGR rooms. Air concentrations of microorganisms and dust and of gaseous pollutants were measured twice weekly. Respiration rate and rectal temperature were monitored throughout the trial at 1430. Behavioral traits of ewes were recorded once per week from 0930 to 1230. Cell-mediated immune response to phytohemagglutinin at d 3, 20, and 40 and humoral response to chicken egg albumin at d 11, 21, 30, and 40 were determined. At d 37, ewes were injected with 2 IU porcine ACTH/kg body weight(0.75) and subjected to 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 weekly for composition and renneting parameters and fortnightly for bacteriological characteristics. Averages of temperature-humidity index values were 78.9, 76.8, and 74.5 in LVR, MVR, and PROGR rooms, respectively. The LVR and MVR treatments resulted in higher NH3 and CO2 air concentrations than PROGR treatment (P < 0.05). The LVR and MVR ewes had higher rectal temperatures than PROGR ewes (P = 0.001). LVR animals also exhibited higher idling compared to PROGR (P < 0.01) and lower feeding times than MVR (P < 0.05) and PROGR animals (P < 0.01). Ewes under the LVR treatment displayed significant lower averages of antibody titers and higher plasma cortisol levels than PROGR (P < 0.01) and MVR ewes (P < 0.05) 60 min after ACTH injection. The LVR treatment resulted in lower yields of milk (P < 0.01) and reduced feed efficiency (P < 0.01) than PROGR treatment. Results suggest that a fan-ventilated system, providing ventilation cycles during the warmest hours of the day and the night at a mean ventilation rate of 66 m3/ewe per hour, may sustain the performance and welfare in lactating ewes raised in warm climates during summer. A ventilation regimen, programmed to operate over upper critical air temperature and relative humidity, may be economically unattractive under these conditions.

The experiment involved 40 mid-lactating Comisana ewes. The animals were housed in straw-bedded pens and assigned to a control group (n = 10) and two test groups of 15 subjects each. Control ewes were never moved from their pen and peers throughout the experiment period, whereas test animals were subjected to either regrouping (RG) or regrouping and relocation (RGRL) three times at weekly intervals (d 7, 14, and 21 of the trial). Three behavioral recordings were conducted after each social and pen exchange over a 6-h period (1000 to 1600). Animals from groups RG and RGRL were bled immediately before each regrouping and relocation and 15 and 60 min afterward. The phytohemagglutinin skin test was performed at d 9, 16, and 23. Milk yield was recorded when the ewes were mixed and moved, and before and after each mixing and moving procedure. Individual milk samples were analyzed for composition, renneting parameters, and bacteriological characteristics; samples with more than 10(6) somatic cells/mL were cultured for mastitis-related pathogens. Control ewes spent more time lying than groups RG (P < 0.001) and RGRL (P < 0.01) and less time in ambulatory activities than RGRL sheep (P < 0.05). Moved and remaining ewes showed a higher number of aggressive interactions than control subjects (P < 0.001 and P < 0.01, respectively). Control sheep showed the highest cell-mediated immune response compared with both moved and mixed ewes (P < 0.001 and P < 0.01, respectively). The RGRL ewes gave lower yields of milk than did the control ewes on the days after the first (P < 0.05) and the second (P < 0.01) change of pen and peers. The RGRL ewes also had a lower fat content (P < 0.05) than control ewes in the milk yielded on the day of the first regrouping and relocation and a lower milk protein content (P < 0.05) on the following day. The RG treatment resulted in the ewes yielding milk with a lower fat content (P < 0.01) on the day of each mixing procedure as compared with controls and in a decreased milk protein content on the days after the first (P < 0.05) and the second regrouping (P < 0.01). One case of subclinical mastitis was recorded in both the RG and RGRL groups, whereas no cases were detected in control group. These findings suggest that regrouping and relocation may cause increased aggression, altered immune responses, and short-term effects on the production performance of lactating ewes. These practices should be performed by attempting to minimize their impact on animal welfare.

Twenty-four Comisana ewes, with no history of mastitis, were included in this study, with eight ewes each in parities 1, 2 and 3. Groups were separately penned on straw litter and ewes were individually checked for yield, composition, renneting properties and bacteriological characteristics of milk from January, when separated from their lambs (50+/-3 days after lambing), to May. Samples with more than 3.5x10(5) somatic cells/ml were cultured for mastitis related pathogens. Milk yield was not significantly affected by parity. The P3 ewes had significantly higher milk protein, casein and fat contents compared to the P1 and P2 ewes. The P3 ewes also had improved renneting ability of milk as compared to the P1 ewes. Quality of milk decreased with lower lactations. The milk of P1 ewes had significantly greater amounts of mesophilic bacteria than the P2 and P3 ewes, as well as higher concentrations of psychrotrophs and total coliforms in their milk with respect to the P3 ewes. Somatic cell counts in milk and the prevalence of subclinical mastitis were not changed by parity, although mastitis infection set in progressively earlier as the number of lactations decreased. These results suggest that ewes in first or second lactation have a less favourable milk secretion status in relation to mastitis than ewes with a higher number of lactations. Milk yield and quality of younger ewes may be improved by offering feed rations that take into account this reduced capacity to mobilise body reserves. Also, most scrupulous control of sanitation of housing, equipment and personnel is necessary.

We reviewed 286 records of patients older than 60 years with upper GI bleeding from 1985 to 1990. The average age was 72.7 +/- 8.9 y. The most common etiologies were: Gastric Ulcer 26%, Erosive gastritis 24%, Esophageal varices 16%, Duodenal Ulcer 13%, Miscellaneous 17% and Unknown causes 3%. Global mortality rate was 38% and the most frequent cause was variceal bleeding in 36%.