The Effect of Brucella abortus on Hydrogen Peroxide and Nitric Oxide Production by Bovine Polymorphonuclear Cells

The effect of Brucella on the generation of microbicidal reactive oxygen and nitrogen metabolites by bovine peripheral polymorphonuclear cells (PMNs) was investigated. The PMNs were recovered from the peripheral blood of control calves and experimental calves previously vaccinated against brucellosis. Significantly larger quantities of NO and H₂O₂ were generated by PMNs from control and experimental calves following activation by heat-killed whole cells or outer membrane protein of Brucella abortus than by non-activated cells (p<0.05–0.01). In contrast, generation of H₂O₂ and NO decreased when PMNs were exposed to the lipopolysaccharide of Brucella. However, the generation of H₂O₂ and NO by activated PMNs from the control and experimental calves did not differ significantly.     

In Vitro Effects of Acellular Milk on the Bactericidal Components of Caprine Polymorphonuclear Neutrophils

The effects of acellular milk on the activity of the microbicidal cationic enzymes of the polymorphonuclear cells of goats were studied in an attempt to explain the phenomenon by which PMN functions fail in mastitis. Assays were undertaken on the myeloperoxidase, lysozyme and elastase activities in a polymorphonuclear cell (PMN) lysate, both in the presence and absence of acellular milk from homologous species. There was a significant decrease (p<0.05) in the activity of lysozyme, myeloperoxidase and elastase in the presence of acellular milk. Superoxide and H₂O₂ production following activation of caprine PMNs by lipopolysaccharide (LPS) was significantly reduced (p<0.05) in the presence of acellular milk. Thus, the microbicidal function of PMNs is significantly impaired in the presence of acellular milk and this may contribute to the development of mastitis in dairy animals.     

The Effect of Activation of Granulocytes on Enzyme Release and Hydrogen Peroxide and Superoxide Production in Buffaloes

Singh, V.K., More, T. and Singh, S., 1997. The effect of activation of granulocytes on enzyme release and hydrogen peroxide and superoxide production in buffaloes. Veterinary Research Communications, 21 (4), 241-247Polymorphonuclear cells kill microorganisms by the stock of antibiotic proteins and peptides stored in their lysosomal granules and have the ability to produce reactive oxygen intermediates (ROI) such as H₂O₂, O ₂ ^– , and HOCl. Since the components involved in the microbicidal functions of buffalo (Bos bubalis) polymorphonuclear cells (PMN) have not been characterized, an assessment was made of the levels of various enzymes, the extent of extracellular release of these enzymes, and also their ability to produce H₂O₂/O ₂ ^– upon activation with opsonized zymosan (OZ) or lipopolysaccharide (LPS). Using GPC-HPLC, OZ was shown to be a more potent secretagogue than LPS, causing a significantly greater release of low-molecular-weight components. Varying levels of the enzymes (myeloperoxidase, lactate dehydrogenase, acid and alkaline phosphatases, -galactosidase, -D-glucuronidase, elastase and lysozyme) were recorded in the buffalo PMN and both the activators (OZ and LPS) caused significant release of all the enzymes except alkaline phosphatase. Both the activators also caused a significant increase in H₂O₂/O ₂ ^– production by the PMN. However, OZ caused a more pronounced activation than LPS. The studies revealed the presence of oxygen-dependent and oxygen-independent microbicidal systems with buffalo PMN, which responded more effectively to zymosan activation.     

Selenium‐enriched Saccharomyces cerevisiae improves growth,antioxidant status and selenoprotein gene expression in Arbor Acres broilers

One hundred and fifty 7‐day‐old Arbor Acres broilers were randomly assigned into five groups: group 1 served as a control that was fed a basal diet without selenium (Se) supplementation; groups 2, 3 and 4 were fed the basal diet supplemented with 0.15, 0.5 and 1.5 mg Se as Se‐enriched Saccharomyces cerevisiae (SSC) per kg of diet; and group 5 was fed the basal diet supplemented with 0.15 mg per kg of Se as sodium selenite (SS). Growth performance, glutathione peroxidase (GP_X) and superoxide dismutase (SOD) activities, total antioxidant capacity (T‐AOC), and malondialdehyde (MDA) content in plasma and liver, and cellular glutathione peroxidase (GP_X‐1) and phospholipid hydroperoxide glutathione peroxidase (GP_X‐4) mRNA levels in liver were determined. Compared with group 1, groups 2–4 exhibited higher body weights (p < 0.05), lower feed/gain ratios, and higher GP_X activities in plasma (p < 0.05) and GP_X and SOD activities and GP_X‐1 and GP_X‐4 mRNA levels in liver (p < 0.05). Compared with group 5, group 2 exhibited higher GP_X activity in plasma on day 21 (p < 0.05). Compared with group 2 and 5, group 3 exhibited lower MDA content in plasma on day 7 (p < 0.05), higher GP_X activity in plasma, SOD activity and GP_X‐1 mRNA levels in liver on day 14 and 21 (p < 0.05), and higher GP_X‐4 mRNA levels on day 14 (p < 0.05). Compared with group 4, group 3 exhibited lower MDA contents in plasma on day 14 (p < 0.05) and in liver on day 21 (p < 0.05), higher T‐AOC in plasma and higher GP_X‐1 mRNA levels on day 14 and 21 (p < 0.05), and higher SOD activity in plasma and higher SOD and GP_X activities in liver on day 21 (p < 0.05). Thus, SSC improves growth and antioxidant status of broilers; the short‐term bioavailability of SS was faster than that of SSC, but the long‐term bioavailability of SSC was greater than SS.     

Ketamine inhibits the phagocytic responses of canine peripheral blood polymorphonuclear cells through the upregulation of prostaglandin E2 in peripheral blood mononuclear cells in vitro

Ketamine has been reported to decrease the immune functions of phagocytes. Previously, we observed that the phagocytic capacity and oxidative burst activity (OBA) of canine peripheral blood polymorphonuclear cells (PMNs) were inhibited by the supernatant from canine peripheral blood mononuclear cells (PBMCs) cultures treated with ketamine. In the present study, we examined whether in vitro treatment with ketamine modulates prostaglandin E₂ (PGE₂) production in PBMCs. Treatment with ketamine or with ketamine-treated PBMCs culture supernatant simultaneously decreased the phagocytic capacity and OBA of PMNs. Ketamine increased PGE₂ production by PBMCs. Recombinant PGE₂ decreased the phagocytic capacity and OBA of PMNs. AH-6809, an E-prostanoid 2 (EP2) antagonist, restored the phagocytic capacity and OBA of PMNs, decreased by either the ketamine-treated PBMCs culture supernatant or recombinant PGE₂. These results suggest that ketamine inhibits the phagocytic responses of canine PMNs, and that this results from the increase in PGE₂ produced by canine PBMCs.     

Ameliorative effects of boron on serum profile in buffalo (Bubalus bubalis) fed high fluoride ration

An experiment was undertaken to evaluate the protective role of boron on the serum profile of buffalo calves fed a high fluoride ration. Twelve male Murrah buffalo (Bubalus bubalis) calves of 6–8 months age, divided into three groups of four calves in each, were fed basal diets and supplemented with sodium fluoride (NaF, 60 ppm) alone or in combination with borax (Na₂B₄O₇.10H₂O, 140 ppm) for 90 days. Boron (B) was added in the ration as borax to make @140 ppm boron (elemental B) on DM basis in treatment II. Dietary F caused a significant (p < 0.05) depressing effect on serum Ca and Zn on day 90 which was improved with B supplementation. However, serum Fe and Cu did not show any significant change on F or F+B supplementation. The serum ALP and phosphorus level were increased significantly (p < 0.05) on F feeding but declined significantly (p < 0.05) when B was fed. The findings suggested beneficial effect of boron on serum minerals and ALP in buffalo calves fed high fluoride ration.     

Effect of tea catechins on regulation of cell proliferation and antioxidant enzyme expression in H2O2‐induced primary hepatocytes of goat in vitro

Tea catechins (TC) are polyphenols that have potent antioxidant activity. The objectives of this study were to determine the effects of TC on antioxidant status of hepatocytes challenged with H₂O₂. Primary hepatocytes of goat were exposed to 1 mm H₂O₂ without or with 5, 50 and 500 μg/ml TC. The cells were harvested at 48 h post‐treatment to determine effects of TC on proliferation, apoptotic features and membrane integrity of cells, and expression of genes and activities of antioxidant enzymes. H₂O₂ exposure caused damage to cells (p < 0.001). A lower concentration of TC (5 μg/ml) displayed a protective effect by inhibiting exorbitant cell proliferation and DNA degradation. Both H₂O₂ exposure and TC pre‐incubation affected expression of antioxidant enzymes at mRNA and protein levels (p < 0.001). The activities of catalase (CAT) (p = 0.027), CuZn‐superoxide dismutase (CuZn‐SOD) (p < 0.001) and glutathione peroxidase (GPx) (p < 0.001) increased with TC pre‐incubation followed by H₂O₂ challenge. Changes of CuZn‐SOD activity induced by H₂O₂ and TC basically paralleled the changes in the corresponding mRNA and protein levels, but the correlation in CAT and GPx expression displayed slightly different patterns at different concentrations of TC. These findings infer that oxidative stress can induce deleterious cellular responses and this unfavourable condition may be alleviated by treatment with TC.     

Enhancement of the increase in intracellular calcium concentration in stimulated neutrophils byMycoplasma hyopneumoniae

Neutrophils isolated from the peripheral blood of pigs free of infection withMycoplasma hyopneumoniae were loaded with a fluorescent indicator (Fura-2) for detection of cytosolic free calcium concentration. The kinetics of the intracellular calcium flux were examined after incubation with or without a pathogenic or a non-pathogenic strain ofM. hyopneumoniae. The basal intracellular calcium concentration was not altered by incubation withM. hyopneumoniae. However, the relative increase in cytoplasmic calcium concentration caused by the addition of opsonized zymosan was significantly (p<0.05) higher in neutrophils incubated withM. hyopneumoniae as compared to neutrophils not incubated withM. hyopneumoniae. Additionally, after zymosan stimulation, the intracellular calcium concentration was greater in neutrophils incubated with a pathogenic strain ofM. hyopneumoniae than in those incubated with a non-pathogenic strain. This suggests thatM. hyopneumoniae alters the signal transduction mechanisms in neutrophils and that this alteration may be related to virulence.Abbreviations [Ca]_i intracellular concentration of calcium - CCU colour changing units/ml - Fura-2/AM pentaacetoxymethyl ester - PBS phosphate-buffered saline, pH 7.2 - TNF tumour necrosis factor     

Activation of equine neutrophils by phorbol myristate acetate or N-formyl-methionyl-leucyl-phenylalanine induces a different response in reactive oxygen species production and release of active myeloperoxidase

Neutrophil (PMN) contribution to the acute inflammatory processes may lead to an excessive generation of reactive oxygen metabolites species (ROS) and secretion of granule enzymes. We compared the effects of either phorbol myristate acetate (PMA) or N-formyl-methionyl-leucyl-phenylalanine (fMLP) in combination with a pre-treatment by cytochalasin B (CB) on the production of ROS and the release of total and active myeloperoxidase (MPO) by isolated equine PMNs. The ROS production was assessed by lucigenin dependent chemiluminescence (CL) and ethylene release by α-keto-γ-methylthiobutyric acid (KMB) oxidation. In the supernatant of activated PMNs, total equine MPO was measured by ELISA and active MPO by the SIEFED (Specific Immunologic Extraction Followed by Enzymatic Detection) technique that allows for the study of the interaction of a compound directly with the enzyme. The stimulation of PMNs with CB-fMLP only modestly increased the release of MPO, but more than 70% of released MPO was active. PMA stimulation markedly increased the production of ROS and release of MPO, but more than 95% of released MPO was inactive. When PMNs were pre-incubated with superoxide dismutase (SOD) prior to PMA activation, the lucigenin enhanced CL, which is linked to the superoxide anion (O₂⁻) production, was much more decreased than KMB oxidation, linked to the hydroxyl-like radical production. The addition of SOD prior to the activation of PMNs by PMA also limited the loss of the activity of released MPO. These results confirm the key role of O₂⁻ generation in the ROS cascade in PMN and reveal its critical role on MPO inactivation.     

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.     

The effect of low levels of dietary cobalt on the chemiluminescence response of polymorphonuclear leukocytes of goats

Twenty ten-week-old newly weaned male Batinah goats were randomly assigned to a control (n = 10) and a treated (n = 10) group and were fed a diet containing 0.1 mg/kg DM cobalt (Co). Goats in the treated group received bi-monthly subcutaneous injections of 2000 μg of hydroxycobalamin. The phagocytic function of the polymorphonuclear leukocytes (PMN) were tested using a luminol-dependent chemiluminescence assay with opsonized zymosan as the phagocytic target. One month after the onset of the experiment PMN from the control group exhibited a significantly (p < 0.05) lower CL response, which continued for the second month. The results of the present study demonstrated that low levels of dietary cobalt leads to an early impairment of phagocytic function. This may at least in part, be an explanation as to why at the field level in Oman young goats fed diets containing low levels of Co appear to be more susceptible to infections.     

Oxygen Concentration and Cysteamine Supplementation During In vitro Production of Buffalo (Bubalus bubalis) Embryos Affect mRNA Expression of BCL‐2, BCL‐XL,MCL‐1, BAX and BID

This study examined the effects of O₂ concentration (5% vs 20%) during in vitro maturation (IVM), fertilization (IVF) and culture (IVC) or supplementation of IVM and IVC media with cysteamine (50 and 100 μm , respectively; IVM, IVF and IVC carried out in 20% O₂), on blastocyst rate and relative mRNA abundance of some apoptosis‐related genes measured by real‐time qPCR in immature and in vitro‐matured buffalo oocytes and in embryos at 2‐, 4‐, 8‐ to 16‐cell, morula and blastocyst stages. The blastocyst rate was significantly higher (p < 0.05) while the percentage of TUNEL‐positive cells was significantly lower (p < 0.05) under 5% O₂ than that under 20% O₂. The mRNA expression of anti‐apoptotic genes BCL‐2 and MCL‐1 was significantly higher (p < 0.05) and that of pro‐apoptotic genes BAX and BID was lower (p < 0.05) under 5% O₂ than that under 20% O₂ concentration at many embryonic stages. Following cysteamine supplementation, the blastocyst rate and the relative mRNA abundance of BCL‐XL and MCL‐1 was significantly higher (p < 0.05) and that of BAX but not BID was lower (p < 0.05) at many stages of embryonic development, although it did not affect the percentage of TUNEL positive cells in the blastocysts significantly. The mRNA expression pattern of these genes during embryonic development was different in 5% vs 20% O₂ groups and in cysteamine supplemented vs controls. At the 8‐ to 16‐cell stage, where developmental block occurs in buffalo, the relative mRNA abundance of BCL‐2 and MCL‐1 was highest under 5% O₂ concentration and that of BAX and BID was highest (p < 0.05) under 20% O₂ concentration. These results suggest that one of the mechanisms through which beneficial effects of low O₂ concentration and cysteamine supplementation are mediated during in vitro embryo production is through an increase in the expression of anti‐apoptotic and a decrease in the expression of pro‐apoptotic genes.     

Selenium and vitamin E increases polymorphonuclear cell phagocytosis and antioxidant levels during acute mastitis in riverine buffaloes

Antioxidant, antiinflammatory and phagocytic activities were studied in milk polymorphonuclear cells (PMNs) isolated from healthy buffaloes (group I) and during clinical mastitis with the treatment of Enrofloxacin alone (group II) and combined treatment with Enrofloxacin and Vitamin E plus selenium (group III). On days 0,3, 8 and 15 the milk Somatic cell count (SCC) were significantly higher in mastitic milk than in milk obtained from healthy buffaloes. In group II SCC decreased significantly on day 3 and day 8, however in group III reduction in SCC was observed on day 3, day 8 and day 15 (P < 0.05). The antiinflammatory activity was evaluated by determining nitrite plus nitrate (NOx) production in the milk PMNs before treatment and on day 8. NOx activity was significantly higher in mastitic milk than from healthy controls, both before and after treatment (P < 0.05). In group II and group III the activity decreased significantly on day 8 (P < 0.05). The Glutathione peroxidase (GSH-Px) activity was estimated in the milk polymorphonuclear cell (PMNs) supernatant. GSH-Px activity was significantly lower in mastitic buffaloes than in healthy controls, both before and after treatment (P < 0.05). In group II levels did not change in response to treatment, whereas in group III levels had increased significantly on day 8 (P < 0.05). The phagocytic activity (PA) (percentage of neutrophil that had phagocytosed 1–6 bacteria) and phagocytic index (PI) (average number of bacteria/ leukocytes counted in 100 cells) of the milk PMNs was significantly lower in mastitic buffaloes (P < 0.05). In group II the PA and PI did not change in response to treatment, whereas in group III both the parameters had increased significantly on day 8 (P < 0.05). The results of the present experiment indicated enhancement of antioxidative and cellular defense and reduction of somatic cell count in the mastitic animals treated with Enrofloxacin and Vitamin E plus Selenium as compared to the Enrofloxacin treatment alone. Hence Vitamin E plus selenium therapy may be added along with the antibiotics for effective amelioration of intramammary infection in buffaloes.     

Therapeutic management of copper deficiency in buffalo heifers: Impact on immune function

To evaluate the magnitude of copper deficiency in Northern India and to examine the various haematobiochemicals, enzymes, vitamins and immune functions affected by copper deficiency, and to identify the parameters which can be of diagnostic importance in copper deficiency, a survey was conducted in 12 districts of Northern India. Significant deficiency of copper was observed in soil, fodder and serum samples of buffalo heifers. Fifty hypocuperaemic buffalo heifers were selected from these areas and were randomly divided into two groups, A and B. The heifers in group A were provided with mineral mixture containing copper sulphate and in group B without copper sulphate. Significant (p < 0.01) improvement in serum ceruloplasmin level was observed within 30 days of treatment, while significant (p < 0.01) improvement in monoamine oxidase and liver cytochrome oxidase was observed at the 60th day of treatment in group A animals. Significant improvement was observed in T₃ and T₄, in the animals of group A within 60 days of treatment. The values of vitamin A and E showed significant (p < 0.01) improvement within 30 days of treatment. The phagocytic activity of neutrophils against Candida albicans significantly (p < 0.01) improved in group A within 60 days of treatment. Similarly, significant improvement in superoxide dismutase activity in red blood cells was observed at the 30th day, and in total leukocytes and whole blood at the 60th day in group A animals. Significant improvement in liver copper level was observed at the 30th day of treatment, while in group B the liver copper was significantly (p < 0.01) depleted at the 60th day of experimentation. Additional copper supplementation improved growth performance significantly in group A.     

Complement Receptor Type 3 (CR3)- and Fc Receptor (FcR)-Mediated Matrix Metalloproteinase 9 (MMP-9) Secretion and Their Intracellular Signalling of Bovine Neutrophils

Complement receptor type 3 (CR3)- and Fc receptor (FcR)-mediated metalloproteinase-9 (MMP-9) secretion and their intracellular signalling of bovine neutrophils were evaluated. Relative density of MMP-9 secreted by neutrophils stimulated with opsonized zymosan (OPZ, stimulant for CR3) was significantly (p < 0.05) increased when the OPZ concentration was increased from 0 to 0.4 mg/ml. Similar results were obtained for neutrophils stimulated with heat-aggregated IgG (Agg-IgG, stimulant for Fc receptor) at concentrations from 0 to 0.40 mg/ml. Preincubation of neutrophils with 1–30 nmol/L wortmannin (phosphoinositide 3-kinase inhibitor) resulted in inhibition of MMP-9 secretion induced by stimulation with OPZ and Agg-IgG in a concentration-dependent manner, 30 nmol/L wortmannin causing complete inhibition. Similarly, preincubation of neutrophils with 0–100 μmol/L genistein (tyrosine kinase inhibitor) also resulted in inhibition of OPZ- and Agg-IgG-induced MMP-9 secretion in a concentration-dependent manner, with 100 μmol/L genistein causing complete inhibition. Significant (p < 0.05) positive correlations were found between MMP-9 and luminal-dependent chemiluminescent response (LDCL) in the case of stimulation with OPZ (r = 0.754) and in the case of stimulation with Agg-IgG (r = 0.728). Our findings suggested that CR3 and FcR play a critical role in production of MMP-9 and may be regulated by intracellular signal transduction, including that by phosphoinositide 3-kinase (PI3K) and tyrosine kinase (TK).     

Evaluation of phagocytic function of canine peripheral polymorphonuclear leucocytes by whole blood chemiluminescence.

Zymosan-induced and luminol-aided chemiluminescence (CL) of whole blood from beagle dogs was estimated for the function of polymorphonuclear leucocytes (PMNs). Whole blood (0.1 ml) was examined directly and results were obtained within 20 min. A phagocytic function of PMNs can be estimated from the peak CL counts and the number of PMNs in a specimen, and the opsonic activity can also be estimated by the peak time showing peak CL after the addition of non-opsonized zymosan. The optimal temperatures to keep diluted whole blood for the CL measurement was around 13 degrees C. Thus, this method offers information concerning the functions of phagocytic cells in whole blood.     

Trans-10, cis-12 conjugated linoleic acid modulates phagocytic responses of canine peripheral blood polymorphonuclear neutrophilic leukocytes exposed to Clostridium difficile toxin B

Trans-10, cis-12 conjugated linoleic acid (t10c12-CLA) has been reported to enhance phagocyte function. Clostridium difficile toxin B (TcdB) has been known to inhibit Ras-homologous (Rho) guanosine triphosphatases (GTPases) which play essential roles in neutrophil immune functions. Here, we examined whether in vitro treatment with t10c12-CLA modulates the filamentous actin (F-actin) polymerization, phagocytic capacity, and oxidative burst activity (OBA) of canine peripheral blood polymorphonuclear neutrophilic leukocytes (PMNs) exposed to TcdB. Treatment with t10c12-CLA, but not linoleic acid, enhanced PMN F-actin polymerization, phagocytic capacity, and OBA, while TcdB suppressed these functions. t10c12-CLA reversed the suppressive effects of TcdB on these PMN functions. t10c12-CLA stimulated F-actin polymerization regardless of whether phagocytosis was stimulated by microspheres but only elevated OBA when microspheres were added. We asked whether the effects of t10c12-CLA were associated with changes in the activation of the Rho GTPase Cdc42. Treatment with t10c12-CLA augmented Cdc42 activity in both TcdB-treated and TcdB-naive PMNs during phagocytosis. Thus, t10c12-CLA up-regulates PMN phagocytic responses attenuated by TcdB. This effect is associated with an increase in actin polymerization and may involve the activation of Cdc42.     

Demonstration of Alternative and Classical Complement Pathway Activity in Colostrum from Buffalo (Bubalus bubalis)

Buffalo colostrum caused lysis of unsensitized red blood cells (RBC) from sheep, goats, rabbits and chickens. RBC from cattle and buffalo were resistant to lysis. That lysis was due to the presence of natural antibodies to these RBC was ruled out since there was no reduction in haemolytic titres even after adsorption with the respective RBC. The addition of EGTA to the diluent had no effect on the haemolytic activity. These findings indicate the presence of alternative complement pathway (ACP) activity in buffalo colostrum. The haemolytic activity of buffalo complement for unsensitized rabbit RBC was reduced to very low levels by heating at 50°C for 45 min. Treatment with zymosan also inhibited the haemolytic activity, while inulin had no effect. The maximum activity of ACP occurred in the presence of 4 mmol/L Mg²⁺ in the diluent. The range of ACP activities in colostrum from buffaloes varied from 4.06 to 8.48 CH₅₀ units/ml. Using a standard system for titrating the classical complement pathway and rabbit red blood cells sensitized with goat haemolysin, the range of complement activity in buffalo colostrum was 4.81–6.77 CH₅₀/ml.     

Effects of Hydrogen Peroxide (H2O2) on Fresh and Cryopreserved Buffalo Sperm Functions During Incubation at 37°C In Vitro

The magnitude of damage to buffalo spermatozoa during incubation with different levels of H₂O₂ was assessed. A total number of 24 ejaculates from four Murrah buffalo bulls were analysed in the study. Each ejaculate was split into two parts (part I and II). Part I was extended in Tris–egg yolk–citrate extender (20% egg yolk:7% glycerol), equilibrated (4 h at 5°C) and cryopreserved in 0.5‐ml French straws and stored in liquid nitrogen. The other part was utilized for fresh semen studies. The sperm in fresh, equilibrated and frozen–thawed semen was separated by centrifugation (1500 g ; 15 min) and were washed with sperm TALP. The sperm cells were re‐suspended in incubation TALP at the rate of 10⁸ sperm cells per millilitre and incubated with 0, 10, 25, and 50 μm H₂O₂ per ml at 37°C. Sperm motility, viability and intact acrosome percentages were assessed at 15‐min intervals up to 60 min of incubation. Lipid peroxidation levels of sperm were assessed at 0 and 60 min of incubation. The results of the experiment revealed that sperm motility decreased drastically during incubation with H₂O₂. Among the different levels of H₂O₂, the 50‐μm H₂O₂‐incorporated group had significantly (p < 0.05) higher malonaldehyde (MDA) level than the other groups. In the 50‐μm H₂O₂‐incorporated group, the MDA levels in fresh, equilibrated and frozen–thawed semen after incubation for 60 min were 961.6 ± 12.7, 991.8 ± 10.3 and 1234.9 ± 9.6 nm per 10⁹ spermatozoa respectively. An inverse relationship was observed between sperm motility, viability, intact acrosome percentages and concentration of H₂O₂ and duration of incubation. The decrease in sperm functions with duration of incubation and concentration of H₂O₂ was significantly (p < 0.05) higher in frozen–thawed than fresh and equilibrated spermatozoa.     

Actin Polymerization: An Event Regulated by Tyrosine Phosphorylation During Buffalo Sperm Capacitation

In the female reproductive tract, the spermatozoa undergo a series of physiological and biochemical changes, prior to gaining the ability to fertilize, that result to capacitation. However, the actin polymerization and protein tyrosine phosphorylation are the two necessary steps for capacitation. In this study, we have demonstrated the actin polymerization and established the correlation between protein tyrosine phosphorylation and actin reorganization during in vitro capacitation in buffalo (Bubalus bubalis) spermatozoa. Indirect immunofluorescence and Western blot techniques were used to detect actin polymerization and tyrosine phosphorylation. The time‐dependent fluorimetric studies revealed that the actin polymerization starts from the tail region and progressed towards the head region of spermatozoa during capacitation. The lysophosphatidyl choline (LPC)‐induced acrosome reaction (AR) stimulated quick actin depolymerization. The inhibitor cytochalasin D (CD) blocked the in vitro capacitation by inhibiting the actin polymerization. In addition, we also performed different inhibitor (Genistein, H‐89, PD9809 and GF‐109) and enhancer (dbcAMP, H₂O₂ and vanadate) studies on actin tyrosine phosphorylation and actin polymerization. The inhibitors of tyrosine phosphorylation inhibit actin tyrosine phosphorylation and polymerization, whereas enhancers of tyrosine phosphorylation stimulate F‐actin formation and tyrosine phosphorylation. These observations suggest that the tyrosine phosphorylation regulates the actin polymerization, and both are coupled processes during capacitation of buffalo spermatozoa.