Heterogeneity of proteolytic enzyme activities in milk samples of different somatic cell count

Milk contains the alkaline proteinase plasmin and lysosomal proteinases; the significance of the latter is ill-defined. The objective of this study was to investigate composition and activities of several different proteolytic enzymes in milk samples of varying somatic cell count (SCC). Increasing milk SCC was correlated with increased plasmin, cathepsin D and cysteine protease activities, with concomitant increases in proteolysis in milk. Addition of plasmin inhibitors confirmed the heterogeneity of proteinase activities in milk, as urea-PAGE analysis of milk samples showed casein hydrolysis in milk after 7 d storage even in samples with inhibitors added; extent and heterogeneity of proteolysis was correlated with milk SCC. Rennet coagulation properties were not significantly correlated with SCC, or activities of measured enzymes. Milk of increasing SCC also exhibited decreased physical stability during incubation of milk at 37 degrees C. Pasteurized milk was more stable than raw milk, suggesting that the enzyme(s) or mechanisms leading to such instability are impaired by pasteurization. Overall, milk has a very heterogeneous proteolytic enzyme population, with a higher significance of non-plasmin enzymes, such as cathepsin D and cysteine proteinases, than perhaps previously recognised.     

Effects of somatic cell count and stage of lactation on the plasmin activity and cheese-making properties of ewe milk

The experiment was conducted from March to July 2002 using 5 intensively managed flocks of Southern Italy. In each flock, 2 groups of 50 ewes were created. The groups were designated LSCC (low somatic cell count [SCC]) when their milk SCC was lower than 500,000/mL and HSCC (high SCC) when their milk SCC was higher than 1,000,000/mL. Bulk milk and whey samples were analyzed for fat, total protein, lactose, casein, and whey protein contents. Renneting properties of milk were also determined. Moisture, NaCl, and nitrogen fractions were determined in fresh cheese curds. In addition, plasmin (PL) and plasminogen (PG) activities in milk and cheese were monitored. The proteolytic activity of plasmin by urea-polyacrylamide gel electrophoresis and the white blood cell (WBC) differentials were determined. The HSCC resulted in higher pH values in milk and in higher moisture and lower fat contents in fresh cheese curds. Moreover, a lower recovery of fat and whey proteins was obtained from the HSCC than from the LSCC raw milk. The crude protein and casein contents were higher in the HSCC than in the LSCC curds during early and midlactation; an opposite trend was observed in late lactation. Plasmin and PG activities underwent more marked fluctuations in the LSCC than in the HSCC curds through lactation. The results of this experiment demonstrate that the PL activity in ewe milk is markedly influenced by the SCC, although SCC is not the only parameter for predicting PL and PG evolution in ewe milk. The LSCC milk resulted in a higher proteolytic potential of Canestrato pugliese cheese curds.     

Environmental factors affecting plasmin activity in milk

A total of 367 milk samples were collected from 43 individual Holstein cows during 1 yr. Samples were analyzed for plasmin activity, total casein, alpha s-casein (alpha s1-casein + alpha s2-casein), beta-casein, kappa-casein, and SCC. Least squares analyses showed that SCC in milk were positively related (r = .62) to plasmin activity. An increase of SCC from 100,000 to 1,300,000/ml was associated with a 2.3-fold increase in plasmin activity (100 vs. 230 x 10(-6) units/ml). Increased plasmin activity was associated with advancing stage of lactation and older cows after appropriate adjustments were made for the effects of milk yield and SCC. Milk samples obtained in fall and winter were higher, but not significantly, in plasmin activity. Plasmin activity was also associated with major casein components and milk pH. Correlations coefficients between plasmin and alpha s-casein, beta-casein, and pH were -.14, -.27, and .19.     

Factors affecting the plasmin-plasminogen system in milk obtained from three Greek dairy sheep breeds with major differences in milk production capacity

The purpose of this study was to evaluate the effect of breed, stage of lactation, and health status of the udder on the plasmin-plasminogen system in ovine milk. A total of 38 ewes were used from 3 breeds [Boutsiko (n = 12), Chios (n = 12), and a synthetic breed (50% Boutsiko, 25% Arta, and 25% Chios, n = 14)] with major differences in their genetic potential with respect to milk yield. Milk samples were collected every 2 wk throughout the lactation period and were analyzed for fat, protein, lactose, and somatic cell count (SCC). In addition, milk plasmin (PL), plasminogen (PG), and plasminogen activator (PA) activities were determined. The Chios breed had the greatest average daily milk yield, the synthetic breed had an intermediate milk yield, and ewes of the Boutsiko breed had the lowest milk yield. Milk samples obtained from the Boutsiko breed had similar PL and PA activities, compared with those obtained from the other 2 breeds. The ratio of PG:PL was less in milk samples from the Boutsiko breed compared with the other 2 breeds, indicative of an increased rate of conversion of PG to PL for this breed. There was no correlation between PL activity and daily milk yield in ewes from all 3 breeds. Activities of PL, PG, and PA were greater in ovine milk with elevated SCC (>300,000/mL) compared with activities in milk with low SCC (<300,000/mL). The ratio of PG:PL was less in the high-SCC group compared with the low-SCC group, which indicates an increased rate of conversion of PG to PL for the high-SCC group. There was a decrease in PG and PA activities as well as in the PG:PL ratio in late lactation milk (mo 5 to 6) when compared with early or mid lactation milk (mo 1 to 4). Thus, the PL-PG system is affected by breed, stage of lactation, and the health status of the udder. No relationship was found between PL activity and daily milk yield in the 3 Greek dairy sheep breeds. Plasmin is not a marker for gradual involution in the Greek sheep breeds studied.     

Effect of milking interval on milk secretion and mammary tight junction permeability in dairy ewes

Twenty-four lactating ewes (Manchega, n = 12; Lacaune, n = 12) in mid lactation were used to assess the short-term effects of different machine milking intervals (4, 8, 12, 16, 20, and 24 h) on milk yield, milk composition, and tight junction (TJ) permeability of mammary epithelia. Milk samples were analyzed for chemical composition, somatic cell count (SCC), and plasmin activity. Plasma lactose, and milk Na and K concentrations were used as indicators of TJ permeability. Milk accumulated linearly for up to 24 h, showing a different rate according to the milk yield of the breed (Manchega, 38 mL/h; Lacaune, 87 mL/h). Milking interval affected milk fat content, which decreased markedly from 4 to 24 h in both breeds, but no differences were observed in milk protein content. The milk contents of casein, true protein, lactose, and total solids also varied according to milking interval. Values of SCC did not vary by breed (175 × 10³ cells/mL, on average), showing the lowest log₁₀ values for the 4-and 24-h milking intervals in both breeds. Plasmin activity in milk increased with milking interval until 20 h of udder filling in both breeds, and was poorly but positively correlated with SCC content (r = 0.39). Plasma lactose increased dramatically after 20 h of milk accumulation, indicating enhanced permeability of mammary TJ. As a result, an increase in Na concentration and in the Na:K ratio, and a decrease in K concentration, were observed in the milk of Manchega ewes. On the contrary, no differences in Na and K concentrations in milk were detected in Lacaune ewes. In conclusion, our results proved that Manchega and Lacaune dairy sheep could maintain high rates of milk secretion during extended milking intervals in the short term, with no effects on udder health and few negative effects on milk yield. Increased TJ permeability, caused by the effect of udder filling, induced changes in milk composition that were more marked in Manchega than in Lacaune ewes.     

Composition, indigenous proteolytic enzymes and coagulating behaviour of ewe milk as affected by somatic cell count

This study was undertaken to assess the effect of somatic cell count in ewe milk on i) composition and hygienic traits; ii) plasmin, cathepsin and elastase activities; iii) leukocyte differential count; iv) renneting parameters. Individual ewe milk samples were grouped according to somatic cell count (SCC) into five classes: SC300 (<300 000 cells/ml), SC500 (from 301 000 to 500 000 cells/ml), SC1000 (from 501 000 to 1 000 000 cells/ml), SC2000 (from 1 001 000 to 2 000 000 cells/ml) and SC>2000 (>2 001 000 cells/ml). Individual milk samples were analysed for pH, chemical composition, microbial features, indigenous proteolytic enzymes, differential leukocyte population, and renneting parameters. Milk yield, lactose, protein, non casein nitrogen, microbial features were affected by SCC level. Plasmin and elastase activities were the highest in samples with more than 1 000 000 cells/ml; plasmin had intermediate values in samples with 300 000 to 1 000 000 cells/ml and the lowest in samples with less than 300 000 cells/ml of milk. Cathepsin D showed significantly lower values in SC300 and SC1000 classes than in SC500, SC2000 and SC>2000 classes. The highest percentages of lymphocyte were found in samples with less than 1 000 000 cells/ml, while the highest levels of polymorphonuclear leukocyte were found in samples with more than 1 000 000 cells/ml of milk. Longer clotting time was found in SC>2000 samples, while reduced clot firmness was observed in SC500 and SC>2000 samples. Results on milk yield and on compositional parameters evidenced an impairment of udder efficiency in ewe milk samples starting from 300 000 cells/ml. Plasmin activity in milk can be considered as a marker of the synthetic and secreting ability of the mammary gland; furthermore plasmin and elastase were consistent with the health status of the udder. Finally cathepsin D played a role in the worsening of renneting properties of ewe milk.     

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.     

Plasminogen activation system in goat milk and its relation with composition and coagulation properties

The activity of plasmin (PL), plasminogen (PG), and plasminogen activator (PA) and their correlation with goat milk components and milk clotting parameters were investigated. Seven late-lactating Saanen goats were used to provide milk samples that were analyzed for PL, PG, and PA activity (colorimetric assay) fat, protein, noncasein nitrogen, nonprotein nitrogen, casein content, and somatic cell count (SCC). Milk clotting parameters (rennet coagulating time = coagulation time; K20 = firming rate of curd; A30 = curd firmness) were measured with a formagraph. Average milk yield and composition were similar to those previously observed in other studies. Plasmin, PG, and PA activity, expressed as units/ml, were, respectively, 20.04 +/- 0.94, 3.21 +/- 0.04, and 1154 +/- 57.61. Plasminogen activity was surprisingly low compared with other species (bovine, ovine), but it was consistent with the high activity of PA. A negative significant correlation was observed between PL and milk casein content. The correlation coefficients between PL and casein/protein ratio and PA and casein/protein ratio were negative and significant. A positive significant correlation was observed between PL and rennet clotting time and PA and rennet clotting time. Also positive was the correlation between PL and K20 and PA and K20. The plasmin activity was negatively correlated with A30. High plasmin and plasminogen activator activity in goat milk appeared to be negatively related with coagulating properties in late lactation, most probably via degradation of casein due to plasmin activity.     

Role of endogenous enzymes in proteolysis of sheep milk

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 α-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°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.     

Quality of milk and of Canestrato Pugliese cheese from ewes exposed to different ventilation regimens

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.     

Long- and short-term effects of omitting two weekend milkings on the lactational performance and mammary tight junction permeability of dairy ewes

The long- and the short-term effects of omitting 2 milkings weekly in early (wk 8 to 14) and mid lactation (wk 15 to 22) were investigated in an experiment conducted with a total of 58 dairy ewes (40 Manchega and 18 Lacaune). Ewes submitted to 2 milking omissions were milked twice daily from Monday to Friday (0800 and 1800 h), and once daily on Saturday and Sunday (1600 and 1400 h, respectively). Individual data were collected for milk yield (weekly), milk composition (biweekly), and somatic cell count (SCC; monthly). Omitting 2 milkings per week in early lactation tended to decrease milk yield in Manchega ewes (−15%), whereas no effects were observed in Lacaune ewes. Averaged milk composition was not modified by milking omissions in either breed. Milking omissions in late lactation did not affect milk yield and milk composition in either breed. The SCC were unaffected by milking omissions in both breeds and in both stages of lactation. A sample of 22 Manchega and 11 Lacaune ewes were used to evaluate the short-term (daily) effects of the 2 milking omissions per week on milk yield and composition, udder health, and tight junction permeability, both in early lactation (wk 12) and in mid lactation (wk 20). Milking omission decreased milk yield, milk fat, and milk lactose contents on the first omission day in both breeds, with losses being more noticeable in early lactation than in mid lactation. Milk protein content and SCC did not vary by effect of the weekend milking omissions. After restoring the twice-daily milking routine on Monday, milk yield showed a compensatory increase that was greater in the large-cisterned than in the small-cisterned ewes, which allowed milk yield to return to Friday values in both breeds. Milk fat content increased during Sunday and Monday, reestablishing Friday values thereafter in both breeds. Weekend milking omissions in early lactation caused tight junction leakiness in both breeds, but mammary epithelia adapted to extended milking intervals when applied successively, recovering their tight state after milking. In mid lactation, the mammary tight junction showed leakiness only in Manchega ewes. In conclusion, 2 milkings per week could be omitted with no negative effects on milk yield, milk composition, and milk SCC values in large-cisterned dairy ewes, as observed in Lacaune and large-cisterned Manchega ewes. Losses in milk yield could be reduced if milking omissions were done from mid lactation in small-cisterned ewes.     

Changes in plasmin-plasminogen-plasminogen activator system in milk from Italian Friesian herds

Changes in plasmin, plasminogen and plasminogen activator (PA) throughout the lactation were investigated in individual milk samples obtained from 32 Friesian cows from four commercial herds located in Northern Italy. Herds were chosen to represent four different, yet typical for Italy, diets. Increased levels of plasmin and PA (P < 0.05) were observed with advancing lactation. Plasminogen peaked during the fifth month of lactation. The increased levels of plasmin during the fifth month of lactation are partly due to increased plasminogen, which reflects increased permeability of mammary epithelium. However, the ratio of plasminogen to plasmin decreased with advancing lactation, suggesting accelerated conversion of plasminogen to plasmin. Major differences were observed between herds with respect to plasmin levels. These differences probably reflect differences in diets and management practices. This could be very important for Northern Italy where most of the milk produced is used for cheese manufacture. Plasmin, PA and somatic cell counts (SCC) were negatively correlated with casein/protein with coefficients of −0.38, −0.43 and −0.40, respectively. A significant correlation existed between PA and SCC (r = 0.50). PA was positively correlated with plasmin (r = 0.49).     

Effects of stage of lactation and time of year on plasmin-derived proteolytic activity in bovine milk in New Zealand

The objective of this study was to determine the effects of stage of lactation (SOL) and time of year on plasmin-derived proteolytic activity in the milk of pasture-fed dairy cows in New Zealand. Four herds of 20 Friesian cows were used, one herd calving in each of January, April, July and October. Cows grazed ryegrass/white clover pasture only, except during June (winter) when all cows received supplementary pasture silage. Milk samples were collected on four occasions during the year (spring, summer, autumn and winter) from each cow in milk, to give a total of three samples per cow (early, mid and late lactation; c. 30, 120 and 220 days after calving, respectively). Milk samples were analysed for plasmin-derived proteolytic activity. There was no effect of either SOL or time of year on plasmin activity and therefore yields of plasmin followed patterns in milk yield (highest in early lactation and in summer). There were effects of both SOL and time of year on plasminogen-derived and total plasmin plus plasminogen-derived activity, both of which were highest in late lactation and in spring. Changes in plasminogen-derived activity and total plasmin plus plasminogen-derived activity due to SOL were not only due to the decrease in milk yield associated with advancing lactation, because enzyme yields were also increased with advancing lactation. Similarly, effects of time of year on plasminogen-derived activity and total plasmin plus plasminogen-derived activity could not be attributed solely to concomitant changes in milk yield, and may be influenced by the variation in the quality and quantity of feed during the year inherent in a pasture-based dairy system. Effects of SOL on proteolytic activity were greater than, and independent of, effects of time of year.     

Plasmin and plasminogen in bovine milk: a relationship with involution?

A total of 774 individual milk samples were collected from 66 Holstein cows between October 1987 and April 1988. Samples were analyzed for plasmin, plasminogen, and SCC. An increase in SCC from less than 250,000/ml to more than 1,000,000/ml resulted in an increase of plasmin, plasminogen, and serum albumin by 105, 74, and 140%, respectively. Plasminogen, plasmin, and serum albumin followed similar trends that are expected for components from blood that gain access to the alveolar lumen through ruptured epithelium caused by mastitis. Increased plasmin is the direct result of this process rather than an increase in activation of plasminogen to plasmin. The plasminogen to plasmin ratio supports this interpretation, being 4.7 at 250,000 SCC/ml and 4.0 when SCC exceeded 1 million/ml. Plasmin and plasminogen concentrations were also increased during lactation to reach peak values immediately before the dry period. However, in this case, ratio of plasminogen to plasmin was 6.55 during early lactation and decreased by half to 3.29 during the latest stage, indicating that considerable activation of plasminogen to plasmin occurred during the latter part of lactation. Mammary epithelium is not compromised at this stage, as shown by low (.8 mg/ml) serum albumin concentration in milk. Two mechanisms responsible for increased milk plasmin include influx of plasmin from blood during mastitis and increased activation of plasminogen as lactation progresses.     

Effect of mastitis on plasminogen activator activity of milk somatic cells.

This study was conducted to examine the effects of mastitis and stage of lactation on plasminogen activator (PA) activity in milk somatic cells. An assay system, which measures the plasmin-mediated hydrolysis of the chromogenic substrate D-valyl-leucyl-lysine p-nitroanilide, was used to assess PA activity present within milk somatic cells. Milk cell associated PA activity was increased (P < 0.05) by 50% in the presence of fibrin fragments. This suggests that milk somatic cells contain tissue PA which, unlike urokinase PA, is preferentially activated in the presence of fibrin fragments. An increase of the milk somatic cell count from < 5 x 10(4) to > 10(6) cells/ml resulted in an 8-fold increase in PA activity per cell. Elevated levels of PA activity were associated with milk somatic cells isolated from mastitic quarters obtained from cows in early (< 4 months in lactation) or late lactation (> 8 months in lactation). We conclude that PA activity is increased during severe mastitic inflammation. Although the physiological function of this enzyme is as yet unclear, we propose that it may be involved in the conversion of plasminogen to plasmin, contributing to the higher levels of plasmin occurring in milk isolated from mastitic quarters.     

Effects of solar radiation and feeding time on behavior, immune response and production of lactating ewes under high ambient temperature

A 6-wk trial was performed with 40 late-lactation Comisana ewes, which were either exposed to or protected from solar radiation and fed either in the morning (EXPM, PROM) or afternoon (EXPA, PROA) during summer in a Mediterranean climate. Behavioral traits of ewes were recorded once per week from 0800 to 2000 h. Rectal temperature (RT) and respiration rate (RR) were measured twice weekly at 1430 h. The phytohemagglutinin (PHA) skin test was performed to induce nonspecific delayed-type hypersensitivity at d 10, 20, and 32 of the experiment. Jugular blood samples were taken from ewes at the beginning and at d 21 and 42 of the experiment. Ewe milk yield was recorded daily. Individual milk samples were analyzed weekly for milk composition, coagulating properties, somatic cell count (SCC) and polymorphonuclear neutrophil leukocyte counts (PMNLC) and every 2 wk for bacteriological characteristics. Solar radiation and the interaction of solar radiation x time of feeding had significant effects on rectal temperatures. EXPM ewes had higher rectal temperatures than EXPA ewes, which in turn exhibited higher RT compared with PROM and PROA ewes. EXP groups also had significantly higher respiration rates than PRO groups. Immune response was lower in EXPM ewes at d 10 and in EXPM, EXPA, and PROM animals at d 20 compared with PROA ewes. Exposure to solar radiation resulted in decreased plasma concentrations of alanine amino-transferase, alkaline phosphatase, potassium, and magnesium, as well as in increased levels of nonesterified fatty acids and aspartate amino-transferase. Milk yield and composition were not changed by exposure to solar radiation and time of feeding, but the EXPM treatment resulted in lower yields of casein and fat and reduced clot firmness compared with the three other treatments. Milk SCC was similar across treatments, but PMNLC was higher in EXPM than in PROM and PROA milk. EXPM animals also had the greatest amounts of total and fecal coliforms and of Pseudomonadaceae as well as the highest number of mastitis related pathogens in their milk. Results suggest that provision of shaded areas can play a major role in helping lactating ewes to minimize the adverse effects of high ambient temperatures on thermal balance and energy and mineral metabolism. Changing the time of feeding to late afternoon may be beneficial to exposed ewes in lowering their heat loads during the warmest hours of the day, thereby reducing the detrimental impact of thermal stress on immune function and udder health.     

Profile of gelatinolytic capacity of raw goat milk and the implications for milk quality

Both endogenous and exogenous proteinases occur in milk, and they can have beneficial or detrimental effects on dairy production. Because the lactation length of dairy goats is shorter and the somatic cell count (SCC) of goat milk is generally greater compared with dairy cows, the objectives of the present study were to investigate the prevalence of major proteinases in raw goat milk, their association with SCC and production stage, and their effects on milk quality. Milk samples were collected from individual goats in consecutive weeks for different durations, covering regular lactation, late lactation, and post-milk stasis. Long-term (monthly) or short-term (weekly) fluctuations of milk fibrinolytic and gelatinolytic capacities of individual goats were revealed chronologically on fibrin and gelatin zymograms, respectively. In a separate trial involving milk samples from 23 goats at random production stages, the percentage of ultracentrifuge force-precipitable casein of total milk protein was calculated to represent milk quality and was assessed to evaluate its correlation with the corresponding proteolytic capacities. The results for regular milk indicate that gelatinase B was more abundant than gelatinase A when they first appeared at SCC of ∼1 × 10⁶/mL. During the last month before milk stasis, both gelatinases A and B were found to be prevalent and prominent in milk regardless of the broad SCC range recorded there. Fibrinolytic activity and the active form of gelatinase A were only regularly detected in post-stasis secretions and were scarce before stasis. The results of the milk quality trial indicate that milk of relatively high proteinase capacity tended to have a low casein ratio. Correlation analysis confirmed a significant relationship between gelatinase capacity of goat milk and production stage, SCC, or casein ratio. It is suggested that an elevation of gelatinolytic capacity of goat milk coincides with an increase in somatic cell number accompanying the extension of lactation length, which is unfavorable for the production of a more desirable quality of goat milk.     

KINETIC PARAMETERS FOR THERMAL INACTIVATION OF PLASMIN

Plasmin (EC 3.4.21.7) is the principal indigenous protease in bovine milk. Kinetic parameters for thermal degradation of plasmin were determined using a miniature continuous flow pasteurizer designed for heat treatment of small quantities of liquid. Plasmin activity was measured using a pH-stat titration, which measures the release of H ⁺ from a caseinate substrate.
Heating milk at various temperatures resulted in significant difference (P > 0.01) in residual plasmin activity and linear decrease in activity at each temperature. The calculated D-values were: 105, 90, 76.5, 62, and 47 s at 72, 78, 85, 92, and 100°C, respectively. The Z-value for plasmin inactivation was estimated to be 77.5°C, and the Arrhenius activation energy was 7.04 kcal/mole.
UHT milk containing plasmin was heated for an equivalent of 5 D inactivation of the enzyme at 100°C. After storage for 30 days at 4°C, no enzyme regeneration was observed.     

Genetic parameters of lactation cell counts and milk and protein yields in dairy ewes

A total of 3231 lactation records of somatic cell counts (SCC), milk yield, and protein percentage for 2379 Spanish Churra ewes from 10 flocks were used to estimate genetic and environmental parameters. Genetic parameters were estimated by REML with a multitrait repeatability animal model. A lactation measure of SCC was obtained as the mean of test day log SCC adjusted for stage of lactation. Heritabilities for SCC, milk yield, and protein percentage were 0.12, 0.24, and 0.17, respectively. The corresponding repeatabilities were 0.35, 0.49, and 0.38. Heritability and repeatability estimates of SCC obtained from this study fell within the range frequently reported for dairy cows. Therefore, as practiced for dairy cattle, future possibilities for sire evaluation to improve udder health status using lactation measures of SCC for dairy sheep are not rejected, although hygienic practices are regarded as more important. Genetic correlations of SCC with milk yield and protein percentage were -0.15 and -0.03, respectively. The genetic correlation between milk yield and protein percentage was -0.47. The low genetic correlations of SCC with milk yield and protein percentage may indicate that breeding decisions to improve milk and protein yields of Churra ewes are not expected to have an effective correlated response in SCC.     

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 α-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.