Calcium homeostasis and its relationship to superoxide production in blood and milk neutrophils of lactating goats.

Polymorphonuclear neutrophils (PMN), which comprise over 70% of the somatic cells in goat milk, are a major cellular component of innate immunity in the goat mammary gland. However, the function of milk PMNs is modified after diapedesis compared to PMNs in blood. As many aspects of PMN activity depend directly on intracellular Ca2+ concentration ((Ca2+)(i)), the present study aimed to determine the changes in Ca2+ homeostasis of milk PMNs from lactating goats compared to autologous blood PMNs, and to examine the significance of these variations to the immuno-competency of milk PMNs. The intracellular Ca2+ store of freshly prepared milk cells was estimated from the elevation of (Ca2+)(i) after ionomycin treatment, which was found to be significantly less than blood PMNs. Replenishment of the intracellular Ca2+ store in milk cells after intracellular Ca2+ depletion by Bapta-AM followed by spiking with 2.5mM Ca2+ for 20min was also compared to that of blood PMNs, showing that after depletion/spiking the intracellular Ca2+ store in milk cells was much less than blood PMNs. The production of superoxide anion (O(2)(-)) in vitro in response to (Ca2+)(i)-dependent or (Ca2+)(i)-independent modulators was used to evaluate the relevance of altered Ca2+ homeostasis on the immuno-competency of milk cells compared to blood PMNs. The results indicated that milk cells produced similarly low levels of O(2)(-) as blood PMNs when treated with ionomycin. However, the amount of O(2)(-) produced by milk cells in response to phorbol 12-myristate 13-acetate (PMA) stimulation, although greater than ionomycin treatment, was significantly less than that of blood PMNs. The capacity for O2(-) production by both cell types in response to PMA reverted to the resting state with use of the protein kinase C (PKC) inhibitor, staurosporine. In conclusion, the current study demonstrated an irreversible shortage of intracellular Ca2+ in the milk PMNs of lactating goats compared to blood PMNs. It also showed that preliminary O(2)(-)production, primed by ionomycin treatment, remained unchanged in milk PMNs, despite the shortage in intracellular Ca2+, but decreased O(2)(-) production capacity, mediated via the PKC pathway, in milk PMN. It is suggested that the defects in Ca2+ homeostasis in milk PMNs of lactating goats is partially attributable for the post-diapedesis functionality modifications.

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 approximately 1 x 10(6)/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.

Contribution of somatic cell-associated activation of plasminogen to caseinolysis within the goat mammary gland.

Functional regression of the mammary gland is partly reflected by proteolysis of milk protein and tissue protein. The involvement of the plasminogen activation system in degradation of milk protein and mammary tissue damage has been demonstrated under inflammatory conditions. In this study, mammary secretion from 23 dairy goats primarily grouped as lactation (milking twice daily) or involution (milking once daily or less) was used to determine the ratio of gravity-precipitated casein to total milk protein (casein ratio) as an index of caseinolysis, and activities of components of plasminogen activation system as well as their expressions on somatic cells. Based on the casein ratio, lactation goats were subcategorized as very active (71.8 +/- 1.0%) or less active (29.9 +/- 1.0%) in mammary function; involution goats were subcategorized as gradual (21.7 +/- 1.0%) or acute (5.9 +/- 0.2%) involution. This result suggests that caseinolysis occurred during regular lactation as well as during involution. On the other hand, activities of components of the plasminogen activation system in mammary secretion were increased along with the decreasing casein ratio, in contrast to the similar activities of their counterparts in circulation throughout various mammary statuses. Correlation analysis between casein ratio and activities of plasminogen activation system of goat milk indicated a significant negative relationship for plasmin (r = -0.64), plasminogen (r = -0.69), and urokinase-type plasminogen activator (uPA; r = -0.78) during involution but not during lactation. As for the cellular components of plasminogen activation system, there was an increase in immunoreactivity on somatic cells toward both monoclonal antibodies of human uPA and human uPA receptor under involution conditions suggesting their upregulation relative to lactation condition. Collectively, these results suggest that plasminogen activation system within the mammary gland differentially contribute to milk caseinolysis along the various stages of goat lactation. Meanwhile, a somatic cell-mediated local elevation of plasmin activity may be committed to extensive caseinolysis during involution.

Sex and genotype dependence on the effects of long-term high environmental temperatures on cellular enzyme activities from chicken organs.

The effects of long term hyperthermia on enzyme levels in the chicken heart and breast muscles, brain, kidney, liver and lung, in relation to sex and degree of feathering, were studied. The enzymes studied were alanine and aspartate amino-transferases, alkaline phosphatase, creatine kinase, lactic dehydrogenase and gamma-glutamyltransferase. Double heterozygote frizzled naked neck and normally feathered male and female broilers were exposed to 24 degrees C (control group) and 32 degrees C (experimental group), for 5 weeks, starting at the age of 3 weeks. The birds were killed, the tested organ removed, homogenized and cell-free supernatant was obtained by centrifugation. Enzyme activities were measured with an autoanalyser and specific activities were calculated. Prolonged heat stress resulted in changes of enzyme activities in all the tissues studied. No significant differences were seen in the cellular enzyme levels from the various organs between male and female birds unexposed to heat stress. Following heat stress, however, greater changes in enzyme levels were seen in the brain, heart muscle and kidney of males compared to females. No significant differences were seen in the cellular enzymes studied in the tissues between the normal and frizzled naked neck chicken. Following prolonged heat stress, there were some differences in the degree of response between the frizzled naked neck and control groups. These differences did not show a consistent or clear pattern indicative of the degree of stress in each of the groups.

The effects of long term high environmental temperature on cellular enzyme activities from different organs.

The effect of long term hyperthermia on enzyme levels in the chicken heart and breast muscles, brain, kidney, liver and lung were studied. Three weeks old chickens were exposed to environmental temperature of 24 degrees C (control group) and 32 degrees C (experimental) for a duration of 5 weeks, after which the birds were sacrificed, organs removed, homogenized and centrifuged at 22,000 g for 30 minutes. Enzyme activities in the supernatant were measured. The following enzymes were analysed: alanine and aspartate aminotransferase, alkaline phosphatase, creatine kinase, lactic dehydrogenase and gamma-glutamyltransferase. Significant changes in cellular enzyme activities were seen in the organs studied. Based on the percentage of changes compared to the controls, large and significant changes were seen in the creatine kinase from heart muscle (mean increase of 328%), aspartate aminotransferase from the brain (mean increase of 148%) and gamma-glutamyltransferase from the kidney (mean increase of 105%). The organs showing the smallest changes were breast muscle and liver.