Comparison of morphology, viability, and function between blood and milk neutrophils from peak lactating goats

The morphological features of blood and milk neutrophils from peak lactating goats were compared using light microscopy, scanning electron microscopy and flow cytometry in order to investigate the cytological changes of neutrophils after migration into the mammary gland. The kinetics of reactive oxygen intermediates (ROI) generation and gelatinase release of blood and milk neutrophils, with or without stimulation of phorbol 12-myristate, 13-acetate ester (PMA), were used to characterize their responses to inflammatory stimuli. Neutrophils isolated from goat milk were highly segmented and contained multi-lobed nuclei. Ultrastructurally, milk neutrophils were more ruffled on the surface compared to blood neutrophils. Approximately 30% of milk neutrophils were undergoing cell death, either necrosis or apoptosis, in contrast to 8% of blood neutrophils. The ROI production of activated milk neutrophils peaked earlier than blood neutrophils, but the duration and the intensity were much less. Neutrophils from both sources augmented the release of gelatinase in response to PMA (1 ng/mL). However, the amount of gelatinase released from milk neutrophils was lower (P < 0.05) than that of blood neutrophils. In summary, more neutrophils become apoptotic and necrotic in the mammary gland, presumably due to spontaneous aging, the process of diapedesis, and the interaction with milk components. Milk neutrophils have impaired functionalities in comparison with blood neutrophils. The information is relevant when studying mammary gland immunity and related diseases, such as mastitis.     

Lowland rainforest avifauna and human disturbance: persistence of primary forest birds in selectively logged forests and mixed-rural habitats of southern Peninsular Malaysia

We compared the composition and structure of primary forest avifauna among primary forests, selectively logged forests and mixed-rural areas (e.g. villages and agricultural areas) of Peninsular Malaysia. We found that forests that were selectively logged at least 30 years ago contained only 73-75% of the 159 species of extant primary forest birds, with an increased proportion of dominant species. We estimated that only 28-32% of the primary forest species utilized the mixed-rural habitat, and that the number of species that bred in the agricultural landscapes might be even lower. The microhabitat of different species most affected their vulnerability to disturbance. Most small, arboreal frugivores and omnivores, and insectivores that fed from tree trunks, showed greater persistence in the mixed-rural habitat than ground dwelling bird species, which were affected most by disturbance. Resource abundance and variables that were closely related to forest disturbance such as the density of large trees, density of dead trees, canopy cover density and shrub volume influenced the distribution of the primary forest birds. Large primary forest reserves and a revision of short-cycle logging regimes (ca. 30 years) are needed if we are to conserve the lowland rainforest avifauna of Peninsular Malaysia and other parts of Southeast Asia.     

Involvement of TNF-α and MAPK pathway in the intramammary MMP-9 release via degranulation of cow neutrophils during acute mammary gland involution

Neutrophil-derived MMP-9 activity is regulated more promptly and efficiently at the level of degranulation than at other levels of regulation. In human neutrophils, degranulation is one of the earliest responses to TNF-α stimulation, which involves protein kinase C and mitogen-activating protein kinase (MAPK) pathways. The level of MMP-9 in mammary secretion of cows increases drastically following milk-stasis, which is partially explained by increases of both neutrophil infiltration and neutrophil degranulation per se. Since MMP-9 represents one of the major remodeling capacities in the mammary gland of cows during early dry period, the current study attempted to explore the involved intracellular mechanisms in the up-regulated MMP-9 secretion. We repeatedly measured on the somatic cells of mammary secretion along the early dry period of cows the expression of TNF-α protein and the phosphorylation of p38 MAPK, ERK, and JNK. Also, cultures of bovine peripheral neutrophils were conducted to examine the mode of short-term MMP-9 secretion in response to TNF-α stimulation and the blocking effects of TNF-α antibody and inhibitors of MAPK pathways. Ex vivo measurements show that conventional cow milk has fully transformed into a neutrophil-abundant, lactoferrin-rich, and high-MMP-9 mammary secretion by d 7 in milk-stasis. No significant (P>0.05) change, however, was found in the expression of TNF-α or the phosphorylation extent of MAPK pathway intermediates on the somatic cells of mammary secretion during the first 3 weeks in milk-stasis. In vitro studies indicate linear increase of short-term MMP-9 release in response to TNF-α stimulation in dosages between 0.1 and 10 ng/ml. In the presence of preparations of d 7-dry secretion of cows, the short-term release of MMP-9 from bovine peripheral neutrophils was significantly (P<0.05) blocked by inhibitor of p38 MAPK but was significantly (P<0.05) promoted by ERK inhibitor while TNF-α antibody or JNK inhibitor exerted no effect. In conclusion, the current ex vivo measurements suggest no apparent association of TNF-α and MAPK pathway with long term intramammary accumulation of MMP-9 during the early dry period of cows, whereas cultures of bovine peripheral neutrophils under a simulated acute involution intramammary environment of cows suggest a role played by TNF-α and MAPK pathways in the short-term MMP-9 release via degranulation.     

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 2.5 mM Ca²⁺ for 20 min 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 (O₂^?) 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 O₂^? as blood PMNs when treated with ionomycin. However, the amount of O₂^? 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 O₂^–production, primed by ionomycin treatment, remained unchanged in milk PMNs, despite the shortage in intracellular Ca²⁺, but decreased O₂^? 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.     

Conservation implications of recent advances in biodiversity–functioning research

Studies have shown that increasing diversity has a positive influence on many ecosystem functions and services, for example, biomass productivity. However, most diversity–functioning studies have derived their conclusions (1) from considering only random species assemblages, (2) from small spatial scales – often micro- and mesocosm experiments, (3) from studying merely a single trophic level, and (4) studies from a small number of biomes dominate. Critics argue that these studies provide little basis to evaluate the consequences for biodiversity loss in the real world. Here we re-consider the latest research focusing on each limitation in turn to highlight the possible lessons for real-world conservation from recent biodiversity–ecosystem function (BEF) research. Tentative general lessons from recent research include: (1) the need to urgently forestall human-induced extinction (i.e., non-random extinction) over large areas, in order to avert large negative functional consequences which may be more pronounced at larger scales and (2) preserve relatively intact communities because biotic interactions across the multi-trophic levels may have a synergistic contribution to the overall functioning of a system. However, considering the complexity of the community dynamics of natural systems, we recommend using natural systems – and understanding the basic physiological features and ecological roles of the species within them – because they implicitly include realistic extinction process, trophic structures and spatial–temporal scales as a useful way of increasing the relevance of future BEF studies to conservation.     

Strategy for the treatment of puerperal metritis and improvement of reproductive efficiency in cows with retained placenta

The objective of this study was to improve the reproductive efficiency of dairy cows with puerperal metritis (PM) subsequent to retained placenta (RP) using a two-step treatment strategy. A total of 188 postpartum cows, aged from 2 to 8 years, were utilised for 2 experiments. In Experiment 1, cows affected with RP/PM were randomly assigned to two treatment groups. Cows in Group A (n = 17) were treated with 600 mg of ceftiofur intramuscularly for 3 days followed by intrauterine lavage with 0.1% chlorhexidine and infusion with 0.5% povidoneiodine, while cows in Group B (n = 16) received two intrauterine infusions, first with 5 g of oxytetracycline and then with 0.5% povidone-iodine. Cows with normal postpartum findings were regarded as the healthy control group (n = 26). Ultrasonographic examination revealed that the ovarian activities including the appearance of a dominant follicle and days to first ovulation of the cows in Group A during the early postpartum period differed from those of Group B (P < 0.05), which coincided with the results of uterine swabbing for bacteriology. In Experiment 2, cows with normal postpartum findings were allocated to Group D (n = 78), which received an ovulation protocol (GnRH - 7 d PGF2α - 48 h hCG - 24 h AI) on day 50 ± 2 postpartum. Cows affected with PM were randomly divided into two groups, Group E (n = 25) combined the treatments applied in Groups A and D, while Group F (n = 26) repeated the treatment administered in Group E except for uterine lavage. The results indicated that the pregnancy rate within 150 days postpartum and the mean days open in Group E (76.0% and 106.3 ± 4.6 days, respectively) were significantly different from those in Group F (38.5% and 137.9 ± 10.9 days, respectively) (P < 0.05). This study suggests that reproductive efficiency could be improved by using the two-step treatment to regulate uterine involution and an early resumption of ovarian function in cows with RP/PM.     

Apoptosis and oxidative stress of infiltrated neutrophils obtained from mammary glands of goats during various stages of lactation

OBJECTIVE: To examine apoptosis in infiltrated neutrophils during involution of mammary glands and compare them with those obtained during late and peak lactation, and to measure oxidative stress and activities of antioxidant enzymes and determine involvement of free radicals in apoptosis of infiltrated neutrophils. SAMPLE POPULATION: Neutrophils from mammary gland secretions of 8 goats at 4 stages (late and peak lactation and 1 and 2 weeks after end of lactation). PROCEDURE: DNA fragmentation was evaluated to characterize apoptosis. Concentration of thiobarbituric acid reactive substances (TBARS) was used to evaluate oxidative stress. Activities of superoxide dismutase and glutathione peroxidase were determined. RESULTS: Neutrophils from secretions obtained after end of lactation of all goats and from late-lactation milk of some goats underwent prominent apoptosis, whereas neutrophils from peak lactation secretions did not. Higher lipid peroxidation and lower antioxidant enzyme activities in neutrophils during involution were observed, compared with those during late and peak lactation. A significant negative correlation existed between TBARS concentrations and antioxidant enzyme activities during the nonlactating period. CONCLUSIONS AND CLINICAL RELEVANCE: Apoptosis is a feature of infiltrated neutrophils during involution of mammary glands in goats. This feature may allow prompt resorption and clearance of infiltrated neutrophils without damaging surrounding tissues. Increased oxidative stress in infiltrated neutrophils from secretions obtained after end of lactation is probably related to a deficiency in antioxidant enzyme activities. Understanding the relationship between apoptosis and oxidative stress will lead to new strategies for manipulating involution and reducing tissue damage.     

Modifications of the defense and remodeling functionalities of bovine neutrophils inside the mammary gland of milk stasis cows received a commercial dry-cow treatment

An appropriate length of milk stasis between two consecutive lactations of dairy cows is crucial for sustainable milk production. This dry period of cows allows extensive remodeling and sufficient cell renewal in mammary gland. Nevertheless, early dry period is one of the most risky stages in cow lactation cycle to intramammary infection. Dry-cow treatment through teats is, therefore, widely practiced at the commencement of milk stasis. Neutrophils are the most abundant cellular components in cow mammary secretion during early dry period, which in turn attribute to the meanwhile elevation of somatic cell counts and matrix metalloproteinase-9 (MMP-9) level. This study used bovine peripheral neutrophils as a cell model to examine the mode of modifications in their defense and remodeling functionalities after infiltration into mammary gland during early dry period. Results indicate a dose-dependent suppression of phorbol 12-myristate 13-acetate-stimulated free radical production and induction of MMP 9 degranulation in bovine peripheral neutrophils exposure to the d 7-dry secretion of cows received dry cow treatment at d 0 in milk stasis. Meanwhile, an enhancement of plasminogen activation and TNF-α shedding on bovine peripheral neutrophils were also observed. These two cellular events might be involved in the functional modifications on infiltrated neutrophils during early dry period. In conclusion, the opposite trend of modifications in the defense and matrix remodeling functionalities of neutrophils inside the mammary gland of cows at early dry period reflect the collaboration of infiltrated neutrophils for promoting extensive glandular remodeling at minimum compromise of local defense during the acute involution period without apparent disturbance by dry cow treatment.     

Hematology and clinical chemistry values of normal and euthymic hairless adult male Dunkin-Hartley guinea pigs (Cavia porcellus)

Hematology and serum chemistry measurements were performed on blood specimens from 12 male Dunkin-Hartley hairless guinea pigs Crl:IAF(HA)BR and 10 haired Dunkin-Hartley male guinea pigs Crl:(HA)BR. Significantly higher activities of alanine aminotransferase, aspartate aminotransferase, amylase, and creatine kinase were observed in the hairless guinea pigs as compared to the haired strain. Alkaline phosphatase activity was found to be lower in the hairless guinea pig. The hairless guinea pigs were found to have serum urea concentrations approximately 46% higher than the normal guinea pig strain. The erythrocytic mean cell volume of the hairless strain was found to be smaller, with a greater hemoglobin content. Hairless guinea pigs were found to have approximately 40% fewer leukocytes with a reversed lymphocyte:neutrophil ratio compared to the haired guinea pigs which had much higher lymphocyte counts.