Melatonin Inhibits Lipopolysaccharide-Induced Inflammation and Oxidative Stress in Cultured Mouse Mammary Tissue.

To determine whether melatonin can protect cultured mouse mammary tissue from lipopolysaccharide- (LPS-) induced damage, we investigated the effects of melatonin on the mRNA and protein levels of proinflammatory cytokines and chemokines in LPS-stimulated mammary tissue in vitro. This study also examined the IgG level in both cultured mammary tissue and the culture medium. In addition, we investigated the potential benefits of melatonin on the expression of antioxidant relative genes following LPS treatment in cultured mammary tissue and evaluated ROS level in the culture medium. The results demonstrate that melatonin inhibited the mRNA expression of TNF-α, IL-1ß, IL-6, CXCL1, MCP-1, and RANTES and the production of these cytokines and chemokines and IgG in LPS-stimulated mouse mammary tissue in vitro. In addition, melatonin increased Nrf2 but decreased iNOS and COX-2 mRNA expression after LPS stimulation. Similarly, the decreased level of dityrosine in the culture medium was increased by treatment with melatonin, while increased nitrite level was suppressed. This study confirms that melatonin inhibited LPS-induced inflammation and oxidative stress in cultured mouse mammary tissue. It might contribute to mastitis therapy while treating antibiotic resistance.

Therapeutic effect of indirubin-loaded bovine serum albumin nanoparticules on ulcerative colitis.

Indirubin is considered to have promising potential in the treatment of ulcerative colitis (UC). However, poor aqueous solubility and low bioavailability limit its clinical application. We produced indirubin-loaded bovine serum albumin nanoparticles (INPs) and characterized their drug encapsulation efficiency, drug-loading capacity, capacity to release indirubin in vitro and short-term physical stability. We also investigated the pharmacokinetics of INPs in mice. We then compared the curative effects of INPs and indirubin against dextran sulfate sodium-induced colitis in mice and 3D cultured biopsies from patients with UC. In the mouse model, the outcomes of INP treatment, including the disease activity index and serous levels of interleukin (IL)-1ß and IL-10, were significantly different from those of indirubin treatment. Similarly, when we administered INPs and indirubin to the ex vivo colonic tissues of patients with UC, the effect of INPs was stronger than that of indirubin for most antioxidant and anti-inflammatory biomarkers. The results of both the animal trial and ex vivo experiment indicate that the therapeutic effect of indirubin was further enhanced by the carrier system, making it a highly promising medical candidate for UC.

The antioxidant effect of triptolide contributes to the therapy in a collagen-induced arthritis rat model.

BACKGROUND: As a chronic autoimmune disease, rheumatoid arthritis (RA) is related to oxidative stress, which may lead to the occurrence and persistence of inflammation in RA. The purpose of this study is to evaluate the potential antioxidant effect of triptolide in collagen-induced arthritis (CIA) rat model. METHODS: We examined the severity of arthritis, levels of local and systemic oxidative stress, periarticular bone erosion and weight of organs in CIA rats treated with triptolide. RESULTS: We found that triptolide decreased the paw thickness and clinical arthritis score, significantly. The mRNA expression and activity of myeloperoxidase and inducible nitric oxide synthase were remarkably decreased in the paws of the CIA rats after triptolide treatment. Triptolide significantly inhibited the levels of nitrite and nitrate in serum, as well as the urinary level of dityrosine. Triptolide treatment also markedly increased bone volume of tibia, but suppressed epiphyseal plate thickness of both femur and tibia. In addition, there was no significant difference in the weight of organs after the therapy, except decreased spleen weight. CONCLUSIONS: These results suggested that the local and systemic oxidative stress was enhanced in the CIA rats and the therapeutic dose of triptolide had a definite antioxidant effect.

Evaluation of three hormonal protocols for anovulatory lactating cows under regulations restricting the use of estrogenic compounds.

When European Union regulations restricted the use of estrogenic compounds in food-producing animals, refined hormonal protocols were no longer applicable for anovulatory cows. However, Ovsynch and its adaptations are routinely and uniformly applied to all cows regardless of ovarian function. To evaluate their efficacy on anovulatory cows, 143, 147 and 144 anovulatory cows received Ovsynch, Presynch and G6G protocols, respectively. In comparison, 150 cyclic cows were bred without using a synchronized protocol. Results showed that cows in the Presynch group had luteolysis responding to the last prostaglandin F2α (PGF2α ) injection greater than the Ovsynch group. The serous progesterone levels at the first gonadotropin-releasing hormone of Ovsych and the last PGF2α injection was greater in the G6G group than the other two hormonal treatment groups. Concentrations of Ca2+ and total protein in cervical mucus in all three hormone-treated groups before artificial insemination (AI) were significantly different from the controls. The G6G group obtained a greater pregnancy rate compared with Ovsynch and Presynch, but significantly less than the controls. For open cows in the Ovsynch group, estrus rate within 24 days after the first AI was significantly less than the controls. In conclusion, the G6G treatment resulted to better reproductive performance in anovulatory cows.

Inline Progesterone Monitoring in the Dairy Industry.

An inline progesterone monitoring system that works automatically and provides real-time physiological information about lactating dairy cows for making farm management decisions is not only a novel tool for scientific research but also improves productivity, food safety, animal well-being, the environment, and the public perception of the dairy industry.

Melatonin Does Not Affect Progesterone Basal Secretion but Suppresses the Luteinizing Hormone Receptor Expression in Granulosa Cells of the Japanese Quail.

The aim of this study was to evaluate the potential effect of melatonin on progesterone production by granulosa cells of the Japanese quail. For in vitro experiments, granulosa cells were isolated from pre-ovulatory follicles (F1-F3) when the F1 follicles were predicted to be either immature or mature (at 3-6 or 18-21 h after oviposition, respectively). Granulosa cells were cultured for 12 hwithor without melatonin concentration gradients of 0.0001-100 µg/mL, thereby averting luteinizing hormone (LH) stimulation. The concentration of progesterone in culture medium was measured using an enzyme immunoassay. The expression of melatonin receptor subtypes in granulosa cells from F1 follicles was detected by reverse transcription-PCR. The LH receptor (LHCGR) mRNA level in cultured granulosa cells of the F1 follicles was analyzed using quantitative real-time PCR. Six quails were used in each of four groups for in vivo experiments. Eachgroup received intraperitoneal injection of melatonin (0.67 mg/kg body weight) or mock-vehicle at 3 or 18 h after oviposition, respectively. The birds were decapitated to collect serum 3 hlater (at 6 or 21 h after oviposition, respectively). The serum progesterone level was also measured using an enzyme immunoassay. We observed that melatonin receptor subtypes (Mel-1a, 1b, and 1c) were expressed in the granulosa cells of the F1 follicles of the Japanese quail. Melatonin suppresses the LHCGR mRNA expression in granulosa cells of F1 follicles but does not affect the basal secretion of progesterone in cultured granulosa cells of the F1-F3 follicles. In addition, melatonin treatment has no influence on the serum progesterone concentration at 6 h post-oviposition, but suppresses progesterone level 21 h after oviposition in the Japanese quail.

Protective Effect of Melatonin on LPS-stimulated Granulosa Cells in Japanese Quail.

The aim of this study was to evaluate the potential of melatonin to protect cultured granulosa cells from the harmful effects of lipopolysaccharide (LPS) in quail. Granulosa cells isolated from Japanese quails were pretreated with or without melatonin (10 or 100 µg/mL) for 12 hand then incubated for 12 hin the absence or presence of 100 ng/mL LPS. The expression of pro-inflammatory cytokines and chemokine was detected by quantitative real-time PCR. The levels of oxidative stress biomarkers (dityrosine and nitrite) were determined by ELISA and the Griess reaction. Cell viability was quantified using an MTT assay. Additionally, the level of progesterone was measured by ELISA. We found that melatonin decreased LPS-induced expression of IL-1ß, IL-6, and IL-8. In addition, melatonin increased the dityrosine level, but suppressed the nitrite level. Finally, melatonin administration increased the viability of LPS-stimulated granulosa cells in vitro. However, progesterone basal secretion was not significantly changed. These results suggest that melatonin protects cultured granulosa cells from LPS-induced inflammatory and oxidative stress damage and provide evidence that melatonin might have therapeutic utility in ovarian follicle infection in Japanese quail.

The anti-inflammatory and antioxidant effects of melatonin on LPS-stimulated bovine mammary epithelial cells.

Mastitis is the most prevalent disease in dairy cattle worldwide and not only causes huge economic losses in the dairy industry but also threatens public health. To evaluate the therapeutic potential of melatonin in mastitis, we examined the ability of melatonin to protect bovine mammary epithelial cells (bMECs) from the harmful effects of lipopolysaccharide (LPS). We found that melatonin inhibited the LPS-binding protein-CD14-TLR4 signaling pathway in bMECs, which had opposing effects on pro-inflammatory and anti-inflammatory mediators. Melatonin decreased LPS-induced expression of pro-inflammatory cytokines, chemokines, and positive acute-phase proteins (APPs), including tumor necrosis factor-α, interleukin (IL)-1ß, IL-6, granulocyte-monocyte colony-stimulating factor, chemokine CC motif ligand (CCL)2, CCL5, serum amyloid A, haptoglobin, C-reactive protein, ceruloplasmin, and α-1 antitrypsin, and increased expression of the anti-inflammatory cytokine IL-1Ra and the negative APP fibrinogen. In addition, melatonin increased dityrosine levels but suppressed nitrite levels by upregulating the expression of Nrf2 and heme oxygenase-1 in the Nrf2 antioxidant defense pathway. Finally, melatonin administration increased the viability of LPS-stimulated bMECs. These results suggest that melatonin protects bMECs from LPS-induced inflammatory and oxidant stress damage and provide evidence that melatonin might have therapeutic utility in mastitis.

Effective embryo production from Holstein cows treated with gonadotropin-releasing hormone during early lactation.

The low efficiency of embryo production in Holstein cows during early lactation presents many challenges for animal production. To improve its efficiency, the outcomes of single GnRH injections 48 hours before each of three cycles of ovum pick up (OPU; weeks 2, 4, and 6) were compared with three cycles of unstimulated OPU (controls; weeks 1, 3, and 5) in 35 Holstein cows during 6 weeks of early lactation (40-80 days postpartum). More total follicle numbers (19.5 vs. 16.0; P < 0.05) but fewer dominant follicles (0.5 vs. 1.4; P < 0.01) were observed by ultrasound, and more cumulus-oocyte complexes were collected in a single OPU in the treatment cycles compared with controls (15.3 vs. 11.5; P < 0.05). The numbers of morphologically "good" cumulus-oocyte complexes graded A and B in the stimulated OPUs were significantly greater than in controls (2.8 vs. 1.7 and 5.8 vs. 4.2, respectively; P < 0.05). Significantly, more oocytes stained positively with brilliant cresyl blue after GnRH treatment compared with the control cycles (13.7 vs. 9.6; P < 0.05). After in vitro fertilization, embryos in the treatment cycles had improved development (P < 0.01) during each developmental stage compared with the controls (9.0 vs. 6.2 two-cell embryos; 4.7 vs. 3.0 four-cell embryos; 3.3 vs. 2.0 morulae; and 3.0 vs. 1.7 blastocysts, respectively). Moreover, there was no significant difference in pregnancy rate of the recipient cows after embryo transfer (57.1% vs. 42.1%; P > 0.05) no matter if the embryos came from the GnRH-treated cycles or not. Thus, GnRH-stimulated OPUs improved the efficiency of embryo production in Holstein cows during early lactation. This novel method for in vitro embryo production should benefit the dairy industry.

A weekly postpartum PGF2α protocol enhances uterine health in dairy cows.

Postpartum uterine health in dairy cows is crucial for the maintenance of good reproductive performance. In order to improve uterine health and reduce puerperal intrauterine infection, 608 Holstein cows received a weekly PGF2α protocol (3 i.m. injections of PGF2α at 7, 14 and 21 d postpartum). For comparison, 593 cows in the control group received injections of sterile saline at the same time. Uterine score at 14 d postpartum, the prevalence of endometritis at 21-27 d postpartum, and subsequent reproduction performance was evaluated. Cows in the treated group exhibited higher tonicity (P<0.05) of the uterus, with less prevalence of endometritis (10.4%, 63/608 vs. 34.6%, 205/593; P<0.001), and required shorter time to the first AI postpartum (67.5±3.4 d vs. 84.4±3.7 d, P<0.05) and to pregnancy (114.5±5.4 d vs. 131.4±5.8 d, P<0.05). In conclusion, the present study demonstrated that uterine health in Holstein cows can be promoted while puerperal infection can be suppressed by this weekly postpartum PGF2α protocol.

Age-associated potency decline in bovine oocytes is delayed by blocking extracellular Ca(2+) influx.

Oocyte aging due to delayed fertilization is associated with declining quality and developmental potential. Intracellular calcium (Ca(2+)) concentration ([Ca(2+)]i) regulates oocyte growth, maturation, and fertilization and has also been implicated in aging. Using bovine oocytes, we tested the hypothesis that oocyte aging could be delayed by reducing [Ca(2+)]ivia blocking the influx of extracellular Ca(2+) or chelating ooplasmic free Ca(2+). After IVM, cumulus-oocyte complexes or denuded oocytes were cultured in medium supplemented with 1-octanol, phorbol 12-myristate 13-acetate, or 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis-acetoxymethyl ester (BAPTA-AM) to manipulate [Ca(2+)]i. Addition of 1-mM 1-octanol increased blastocyst development rates in the cumulus-oocyte complexes aged for 6 hours by IVF and for 6, 12, and 24 hours by parthenoactivation, and this effect was independent of the presence of cumulus cells. The intracellular levels of ATP, Glutathione, and Glutathione disulfide were not affected by 1-octanol, but [Ca(2+)]i was significantly decreased. When oocytes were cultured in Ca(2+)-free medium for 12 hours, the blastocyst development rate was greater and the beneficial effects of 1-octanol on oocyte aging were abolished. However, when the medium was supplemented with phorbol 12-myristate 13-acetate, [Ca(2+)]i increased and the blastocyst development rate decreased. Moreover, BAPTA-AM reduced [Ca(2+)]i and increased blastocyst development rates after IVF or parthenoactivation. We conclude that the age-associated developmental potency decline was delayed by blocking the influx of extracellular Ca(2+) or reducing ooplasmic free Ca(2+). 1-Octanol, BAPTA-AM, or Ca(2+)-free medium could be used to lengthen the fertilization windows of aged bovine oocytes.