Pre-treatment but not co-treatment with vitexin alleviates hyperthermia induced oxidative stress and inflammation in buffalo mammary epithelial cells.

This study investigated if in vitro supplementation of vitexin could mitigate the adverse effects of hyperthermia on buffalo mammary epithelial cells (BuMECs). Immortalized BuMECs were divided into seven groups (n = 3): (1) a negative control group at 37 °C; (2) BuMECs exposed to heat stress as a positive control at 42 °C for 1 h; (3-7) heat stressed BuMECs pre-treated or co-treated with different concentrations of vitexin (5 µM, 10 µM, 20 µM, 50 µM, and 100 µM), respectively. Hyperthermia was induced by exposing the cells to 42 ºC for 1 h. For the pre-treatment experiment, BuMECs were treated with vitexin for 2 h before hyperthermia exposure. For co-treatment, vitexin was added simultaneously with hyperthermia for 1 h. Subsequently, the cells were allowed to recover for 12 h at 37 °C. Results showed that pre-treatment with vitexin was more effective than co-treatment in protecting BuMECs from hyperthermia in a dose-dependent manner, with higher concentrations (50 µM and 100 µM) being the most effective. Pre-treatment with vitexin maintained cellular viability and prevented inflammation by inducing the expression of the anti-apoptotic gene (BCL-2) and reducing the expression of the pro-apoptotic gene (Bax) and pro-inflammatory mediators (IL-1ß, IL-6) in heat-stressed BuMECs. Pre-treatment with vitexin reduced oxidative stress and induced thermotolerance by increasing the expression of antioxidants mediators such as SOD, GPx and CAT at mRNA and protein levels, and modulating the expression of heat shock proteins. The findings suggest that vitexin has the potential as a therapeutic agent to protect the mammary gland from the negative impact of hyperthermia in dairy cows.

Curcumin induces thermotolerance by reducing oxidative stress, apoptosis, and inflammation in buffalo mammary epithelial cells under heat shock conditions.

The epithelial cell is the main basic unit of the udder in which milk synthesis takes place. Curcumin is well known for its antioxidant, anti-apoptotic, and anti- inflammatory properties. The present study was performed to test whether in vitro curcumin supplementation can alleviate the unfavorable impact of hyperthermia on buffalo mammary epithelial cells (BuMECs). The spontaneously immortalized BuMECs were divided into 7 groups (n = 9); 1) unstressed BuMECs (negative control, 37 °C); 2) BuMECs exposed to hyperthermia without curcumin treatment (positive control); 3-7) BuMECs cultured with different concentrations of curcumin (5, 10, 20, 40 and 60 µM), respectively, followed by hyperthermic exposure (42ºC) for 1 h and then returned to 37ºC. Changes in viability (MTT assay), proliferation (BrdU colorimetric immunoassay) and concentrations of antioxidant enzymes, CAT, and SOD (ELISA) of BuMECs were recorded. The gene expression study was performed using qRT-PCR. Lower concentrations of curcumin (5, 10 µM) maintained viability, enhanced proliferation, and content of antioxidant enzymes of heat stressed BuMECs. Curcumin induced thermotolerance and antioxidant status by upregulating the expression of antioxidants genes, anti-apoptotic genes and heat shock proteins in heat stressed BuMECs compared to the positive control group. Besides, curcumin reduced apoptosis and inflammation in BuMECs exposed to hyperthermia by downregulating the expression of genes and transcriptional factors associated with apoptosis and inflammatory immune response. The results reveal the potential roles of curcumin in eliminating the negative impact of hyperthermia on BuMECs by regulating the pathways of apoptosis, inflammation, and oxidative stress.

Astaxanthin inhibits cytokines production and inflammatory gene expression by suppressing IκB kinase-dependent nuclear factor κB activation in pre and postpartum Murrah buffaloes during different seasons.

AIM: We examined regulatory function of astaxanthin on mRNA expression of nuclear factor κB (NF-κB) p65, interleukin-6 (IL-6), tumor necrosis factor alpha (TNF-α), and interferon gamma (IFN-γ) in peripheral blood mononuclear cells in pre and postpartum Murrah buffaloes during summer (temperature-humidity index [THI]=86; relative humidity [RH]=24) and winter (THI=58.74; RH=73) seasons. MATERIALS AND METHODS: A total of 32 Murrah buffaloes apparently healthy and in their one to four parity were selected from National Dairy Research Institute herd and equally distributed randomly into four groups (control and supplemented groups of buffaloes during summer and winter season, respectively). All groups were fed according to the nutrient requirement of buffaloes (ICAR, 2013). The treatment group was supplemented with astaxanthin at 0.25 mg/kg body weight/animal/day during the period 30 days before expected date of calving and up to 30 days postpartum. RESULTS: There was downregulation of NF-κB p65 gene in all the groups. NF-κB p65 mRNA expression was lower (p<0.05) in treatment than control group from prepartum to postpartum during summer, while mRNA expression was low only on day 21 after calving during winter season. The mRNA expression of IL-6, TNF-α, and IFN-γ was lower (p<0.05) in treatment than a control group of buffaloes during summer and winter seasons. The mRNA expression of NFkB p65, IL-6, TNF-α, and IFN-γ was higher (p<0.05) in summer than in winter seasons. CONCLUSION: The xanthophyll carotenoid astaxanthin a reddish-colored C-40 compound is a powerful broad-ranging antioxidant that naturally occurs in a wide variety of living organisms, such as microalgae, fungi, crustaceans, and complex plants. Astaxanthin blocked nuclear translocation of NF-κB p65 subunit and IκBα degradation, which correlated with its inhibitory effect on IκB kinase (IKK) activity. These results suggest that astaxanthin, probably due to its antioxidant activity, inhibits the production of inflammatory mediators by blocking NF-κB activation and as a consequent suppression of IKK activity and IκB-degradation.

Effect of zinc and vitamin E supplementation on hormones and blood biochemicals in peri-partum Sahiwal cows.

Thirty-two advanced pregnant multiparous Sahiwal cows were used to study the effect of additional zinc (Zn) and vitamin E (VE) supplementation on hormonal and biochemical changes. Cows were randomly assigned to four groups and fed a basal diet of compounded concentrate, berseem fodder, and wheat straw in a ratio of 60:20:20. The groups were: (1) the basal diet with no supplement (control treatment); (2) the basal diet supplemented with 60 mg/kg DM/cow daily of Zn (Zn treatment); (3) the basal diet supplemented with 1000 IU/cow daily of vitamin E (VE treatment); and (4) the basal diet supplemented with a combination of 60 mg Zn/kg DM/cow and 1000 IU vitamin E/cow/d (Zn + VE treatment). Blood samples were collected on -60, -45, -30, -15, -7, -3, 0, 3, 7, 15, 30, 45, 60, 90, and 120 d in relation to expected date of calving and were analyzed for endocrine variables and biochemical changes. Plasma concentrations of leptin, insulin, insulin like growth factor-1 (IGF-1), triidothyronine (T3), and tetraiodothyronine (T4) were decreased toward calving and observed lowest (P < 0.05) on 3 d post-partum. However, plasma levels of growth hormone (GH) and cortisol increased toward calving and were found highest (P < 0.05) on 3 d post-partum. Pre-partum concentrations of leptin and IGF-1 were higher (P < 0.05) than its respective concentration observed during post-partum. Post-partum concentrations of GH and cortisol were higher (P < 0.05) than its respective pre-partum concentration. Pre-partum concentrations of urea, triglycerides, Zn, and VE were higher (P < 0.05) and total cholesterol and HDL cholesterol were lower than its values observed in post-partum among all the groups. Treatments had significant (P < 0.05) effect on plasma hormonal levels and levels of Zn and VE but no effect on biochemical attributes. Cows fed on diet supplemented with Zn + VE had highest (P < 0.05) pre as well as post-calving concentrations of leptin (6.38 vs 5.01 ng/ml), insulin (1.39 vs 1.33 ng/ml), GH (9.29 vs 13.72 ng/ml), IGF-1 (14.55 vs 12.59 nmol/l), T3 (1.45 vs 1.40 ng/ml), T4 (32.44 vs 31.79 ng/ml) whereas as lowest concentration of cortisol hormone (3.05 vs 3.44 ng/ml). Cows supplemented with combination of Zn and VE showed minimum decline in plasma concentration of leptin, insulin, GH, IGF-1, T3, and T4, and minimum increase in cortisol concentration. In conclusion, dairy cows around parturition faces various endocrine and biochemical alterations and supplementation of Zn in combination with VE can ameliorate adverse effect of calving stress by maintaining circulatory concentration of hormone and biochemicals towards the basal levels.

Inorganic zinc supplementation modulates heat shock and immune response in heat stressed peripheral blood mononuclear cells of periparturient dairy cows.

Thermal stress in India is one of the major constraints affecting dairy cattle productivity. Every attempt should be made to ameliorate the heat and calving related stress in high producing dairy cows for higher economic returns. In the current study, inorganic zinc was tried to alleviate the adverse effects of thermal stress in periparturient cows. Twelve cows, six each of Sahiwal and Karan Fries (KF) in their second parity with confirmed pregnancy were chosen for the experiment. The blood samples were collected periparturiently on three occasions viz. -21, 0 and +21 days relative to calving. The in vitro study was conducted after isolating peripheral blood mononuclear cells (PBMC) from whole blood. The cultured PBMC were subjected to three different levels of exposures viz. 37°C as control, 42°C to induce thermal stress and 42°C + zinc to ameliorate the adverse effects of high temperature. Heat shock lead to a significant (P<0.05) rise in the level of heat shock proteins (HSP). HSP was more on the day of calving as well. KF showed more HSP concentration than Sahiwal breed indicating the heat bearing capacity of later. Zinc treatment to thermally stressed PBMC caused a fall in the HSP concentration in both the breeds during periparturient period. Moreover, heat stress increased significantly (P<0.05) the Interleukin 6 (IL-6) concentration which declined upon zinc supplementation to PBMC. IL-6 levels decreased periparturiently. Heat and calving related stress caused a fall in the IL-12 levels which increased significantly (P<0.05) with zinc supplementation. These findings suggest that zinc supplementation attenuates the HSP response and augments immunity in PBMC of periparturient dairy cows. The study could help to alleviate the heat stress and potentiate immunity by providing mineral supplements in periparturient dairy cattle habituating tropics.

Effect of in vitro zinc supplementation on HSPs expression and Interleukin 10 production in heat treated peripheral blood mononuclear cells of transition Sahiwal and Karan Fries cows.

The changing climatic scenario with apprehended rise in global temperature is likely to affect the livestock adversely vis-à-vis production and reproduction. This has prompted more focus in addressing the unfavorable effects of thermal stress in livestock system. Presuming that the trace element zinc is indispensible for cellular antioxidant system and immune function, the present study was designed to investigate the effect of zinc treatment on heat stress alleviation and immune modulation in peripheral blood mononuclear cells (PBMC) of indigenous and crossbred transition cows. Twelve cows, six each of Sahiwal and Karan Fries (KF) in their second parity with confirmed pregnancy were selected for the experiment. The blood samples were collected at -21, 0 and +21 days in relation to expected date of calving. The experiment was carried out in vitro after isolating PBMC from whole blood. The 48h cultured PBMC were subjected to assorted levels of exposures viz. 37°C, 42°C to impose heat stress and 42°C+zinc to alleviate heat stress and modulate immunity. The PBMC viability was 86%, 69% and 78%, respectively. The mRNA expression of heat shock proteins (HSP 40, 70 and 90α) and Interleukin-10 (IL-10) production varied between the two breeds vis-à-vis days and levels of exposure. The mRNA expression of HSP40 and HSP70 was significantly (P<0.05) higher in Karan Fries than the Sahiwal cows. Both the breeds showed maximum expression of HSP on the day of parturition, more so in KF than Sahiwal. There was a significant (P<0.05) difference in the HSP mRNA expression at different levels of exposure. Zinc treatment to heat stressed PBMC caused a significant (P<0.05) down regulation of HSP. For immune status, anti-inflammatory cytokine, IL-10 in the culture supernatant was accessed. The IL-10 was significantly (P<0.05) higher in Karan Fries (168.18±14.09pg/ml) than the Sahiwal cows (147.24±11.82pg/ml). The IL-10 concentration was highest on the day of calving. Zinc treatment reduced the IL-10 concentration. From the study, it could be concluded that the zinc supplementation in heat stressed PBMC can ameliorate thermal stress and modulate immune response which can act as a model for reducing heat stress during the periparturient period in tropical livestock.

Heat shock protein 70, oxidative stress, and antioxidant status in periparturient crossbred cows supplemented with α-tocopherol acetate.

The study was conducted to investigating the effect of α-tocopherol acetate on heat shock protein 70 (Hsp70), oxidative stress, and antioxidant status during periparturient period in medium body condition score crossbred cows. Twenty crossbred Karan Fries cows with confirmed pregnancy were selected 2 months before expected date of calving. The cows were randomly distributed in to two groups: 10 cows were kept as control and 10 were supplemented with α-tocopherol acetate during dry period for 2 months. Blood samples were collected at -20, -10, -5, 0, 5, 10, and 20 days in relation to the expected date of calving. Superoxide dismutase, catalase, and total immunoglobulin were significantly higher (P < 0.01) in treatment as compared to control cows. Heat shock protein 70 and thiobarbituric acid reactive substance levels were significantly lower (P < 0.01) in the treatment cows than their counterpart. Treatment with α-tocopherol acetate during dry period resulted in reduced oxidative stress, heat shock protein Hsp70 levels, improved antioxidant, and improved immunity status indicating beneficial effect of α-tocopherol acetate treatment.

Recalcitrant Trichomonas vaginalis infections successfully treated with vaginal acidification.

BACKGROUND: Recalcitrant vaginal trichomoniasis is extremely distressing for patients and frustrating for physicians because there are no current guidelines for treatment. Numerous studies have shown that an increase in vaginal pH creates a better environment for the growth of Trichomonas vaginalis. We describe two patients with recalcitrant trichomoniasis who were successfully treated using vaginal acidification. CASES: The first patient with trichomoniasis had a severe reaction to metronidazole, but the infection subsequently resolved after treatment with a combination of boric acid and clotrimazole. The second patient with resistant trichomoniasis had been treated unsuccessfully with multiple courses of metronidazole but was treated successfully with vaginal acidification using boric acid. CONCLUSION: A process of vaginal acidification resulted in resolution of recalcitrant Trichomonas vaginalis in two patients.