Influenza del metabolismo sulle caratteristiche produttive e riproduttive della bufala Mediterranea Italiana

Italy can be considered a leading country for both genetic improvement and breeding techniques in buffalo species. However, few information are actually available on physiology of this species and particularly those for high producing dairy buffaloes maintained in intensive and semi-intensive breeding. Therefore, it is essential to carry out new studies aimed to increase metabolism knowledge in this species. The purposes of this doctoral thesis were to evaluate: 1) the results of the application of the out of season breeding mating technique and the metabolic status of buffaloes undergone milk recording; 2) the metabolic changes that occur throughout the lactation and in different climate conditions; 3) the possibility of using some parameters assessed in the milk as risk indexes for metabolic diseases; 4) the influence of milk production and some metabolites on pregnancy. Two trials were carried out in the experimental design. The main aim of Experiment 1 was to verify milk production and Beta-Hydroxybutyrate (BHBA) levels in primiparous and pluriparous buffaloes undergone milk recording. Experiment 2 was carried out simultaneously in 8 commercial buffalo farms located in the area of buffalo mozzarella cheese DOP production. The feedstuffs utilized for animal feeding were collected in each farm and analyzed for chemical composition. Milk yield was recorded monthly and two milk samples (during morning and afternoon milking) were collected to evaluate: fat, protein, lactose, urea, true protein, casein, CF, H-index, cryoscopic index, acetone, BHBA and fat characteristics (satured and unsatured fats). Furthermore, 40 buffaloes with different days in milk (5-50 days; 50-120 days; 120-200 daysand 200-270 days) were selected in each farms to analyze metabolic profile and its relationship with metabolites in milk. Therefore, these buffaloes underwent blood sampling two months apart when milk recordings were carried out to analyze the metabolic profile (see below). Throughout the experimental, period 3 milk tank sampling were performed in 6 farms to perform chemical analysis and cheese yield. Finally, the influence of metabolic status on reproductive performance was assessed in each farm, mainly looking at oocyte and embryo development. Therefore, a representative number of buffaloes in each farm underwent synchronization of ovulation and artificial insemination. The results of Experiment 1 demonstrated that the out of season breeding mating technique is correctly applied in primiparous buffaloes, while a higher seasonal pattern can be observed in pluriparous counterparts. Furthermore, BHBA levels were higher at both the beginning and the end of lactation, compared to the mid lactation period. The analysis of urea levels in milk support the evidence that protein requirements were not satisfied or exceeded the requirements in about 50% of cases. In Experiment 2, it was observed that the number of days in milk and the season influence energy metabolism through a lower energy intake, higher metabolic strain (for both the end of lactation and cold periods) and higher milk yield. These buffaloes underwent an excess of fat mobilization, that was the main reason for milk qualitative characteristics worsening and lower yield in mozzarella cheese. Furthermore, the lower dry matter intake and the lipomobilization, recorded at the beginning of lactation caused a change in fat characteristics, as demonstrated by the evidence that buffaloes at lower than 50 days in milk showed a significantly (P<0.01) increase of saturated fatty acids concentration (10.52 ± 0.09 vs. 4.78 ± 0.10, 5.41 ± 0.10 and 5.55 ± 0.10, in 5-50 days; 50-120 days; 120-200 daysand 200-270 days, respectively) together with a decrease of monounsaturated counterparts (1.76± 0.04 vs. 1.88 ± 0.04, 2.40 ± 0.04, and 2.74 ± 0.04 in 5-50 days; 50-120 days; 120-200 daysand 200-270 days, respectively). This was also accompanied by low reproductive efficiency. Furthermore, milk and blood parameters were compared during cold and hot season: the low temperatures and the high thermal excursion recorded during the transitional period (middle of December – end of February) caused lower milk yield (10.96±0.20 vs. 10.16±0.32; P<0.05, in warm and cold period, respectively), lactose (4.94±0.01 vs. 4.88±0.01; P<0.01) and fat / protein ratio (1.85±0.02 vs. 1.75±0.02; P<0.01). The worse climatic conditions recorded were responsible for higher energy requirements, which caused higher fat mobilization demonstrated by milk acetone levels (0.28±0.01 vs. 0.50±0.01; P<0.01), blood NEFA (0.13±0.01 vs. 0.24±0.01; P<0.01) and BHBA levels (0.44±0.01 vs. 0.47±0.01; P<0.05). The statistical analysis of the data also demonstrated a negative correlation between BHBA levels (r =-0.57; P<0.01) and cheese yield. Multiple logistic regression showed that the increase of BHBA levels lowered pregnancy probability (odds ratio = 0.406; P<0.05). In particular, pregnancy probability was lower than 50% in buffaloes with BHBA values higher than 0.25 mmol/L. In conclusion, the metabolic condition due to higher milk yield, climatic conditions and milk fat synthesis caused an increase of metabolites in both blood and milk. Fat mobilization worsened also cheese yield, animal welfare and reproductive efficiency. The possibility of using some metabolites assayed in the milk as risk indicators for some metabolic pathologies may represent a new border to increase animal welfare and improve buffalo production and reproduction

Pregnancies following Protocols for Repetitive Synchronization of Ovulation in Primiparous Buffaloes in Different Seasons

Primiparous buffaloes were tested in two periods of the year characterized, by either low or high reproductive efficiency. They were subjected to two protocols for synchronization of ovulation: (i) Ovsynch (OV) and (ii) progesterone based (P4) treatment. After calving, the animals underwent a series of four cycles of re-synchronization protocols. The season did not affect pregnancy rates when the results of the two treatments were pooled together with regard to the first synchronization protocol, followed by AI. Pregnancy rates were similar during the low breeding season (50.3% vs. 57.4% in OV and P4, respectively), but different during the high breeding season (50.4% vs. 67.7% in OV and P4, respectively; p = 0.000). Logistic regression confirmed a significant effect of treatment and season interaction on pregnancy (p = 0.003). Following re-synchronization, a treatment by season interaction was detected during the low breeding season (odds ratio = 2.233), in favor of P4. Finally, a survival analysis showed a better response of animals subjected to P4 treatment from the second AI onward. In conclusion, the pooled data of pregnancy rates from both treatments between seasons are not different following AIs. Better results, though, were obtained from the implementation of P4 treatment, and are recorded in a season-fashioned mode when the comparison is made following first or cumulative AIs

Laparoscopic gynecological surgery under minimally invasive anesthesia: a prospective cohort study

The purpose of this study is to assess the feasibility and the perioperative outcomes of laparoscopic gynecological surgery in regional anesthesia (RA) from the point of view of the surgeon, anesthesiologist and patient. This is a prospective cohort study comprising sixty-six women planned to undergo gynecologic laparoscopy surgery for benign pathology at tertiary care gynecolgical center of the University Federico II of Naples. Women were assigned, according to their preference, to either RA (Group A) or general anesthesia (GA) (Group B). Surgical, anesthesiologic and postoperative recovery data were recorded. Postoperative pain was considered as the primary outcome. Secondary outcomes included mobilization, length of hospital stay, global surgeons and patient satisfaction, intraoperative pain assessment in Group A. Immediate postoperative pain was significantly lower in Group A 0 vs 2 (p &lt; 0.001), with no significant differences at 24 h. The secondary outcome demonstrated early patient's mobilization (p &lt; 0.001) as well as early discharge (p &lt; 0.001) and greater patient's satisfaction for the Group A. In these patients, a maximum pain score of 3 points out of 5 was recorded through the entire surgery. RA showed to decrease the impact of surgical stress and to guarantee a quicker recovery without compromising surgical results. Although several surgical approaches can be employed to treat different conditions, RA technique could be a viable option for well-selected patients affected by gynecological diseases

The Bile Acid Receptor GPBAR-1 (TGR5) Modulates Integrity of Intestinal Barrier and Immune Response to Experimental Colitis

BACKGROUND: GP-BAR1, a member G protein coupled receptor superfamily, is a cell surface bile acid-activated receptor highly expressed in the ileum and colon. In monocytes, ligation of GP-BAR1 by secondary bile acids results in a cAMP-dependent attenuation of cytokine generation. AIMS: To investigate the role GP-BAR1 in regulating intestinal homeostasis and inflammation-driven immune dysfunction in rodent models of colitis. METHODS: Colitis was induced in wild type and GP-BAR1(-/-) mice by DSS and TNBS administration. Potential GP-BAR1 agonists were identified by in silico screening and computational docking studies. RESULTS: GP-BAR1(-/-) mice develop an abnormal morphology of colonic mucous cells and an altered molecular architecture of epithelial tight junctions with increased expression and abnormal subcellular distribution of zonulin 1 resulting in increased intestinal permeability and susceptibility to develop severe colitis in response to DSS at early stage of life. By in silico screening and docking studies we identified ciprofloxacin as a GP-BAR1 ligand. In monocytes, ciprofloxacin increases cAMP concentrations and attenuates TNFα release induced by TLR4 ligation in a GP-BAR1 dependent manner. Treating mice rendered colitic by TNBS with ciprofloxacin and oleanolic acid, a well characterized GP-BAR1 ligand, abrogates signs and symptoms of colitis. Colonic expression of GP-BAR1 mRNA increases in rodent models of colitis and tissues from Crohn's disease patients. Flow cytometry analysis demonstrates that ≈90% of CD14+ cells isolated from the lamina propria of TNBS-treated mice stained positively for GP-BAR1. CONCLUSIONS: GP-BAR1 regulates intestinal barrier structure. Its expression increases in rodent models of colitis and Crohn's disease. Ciprofloxacin is a GP-BAR1 ligand

Natural iminosugar (+)-lentiginosine inhibits ATPase and chaperone activity of Hsp90

Heat shock protein 90 (Hsp90) is a significant target in the development of rational cancer therapy due to its role at the crossroads of multiple signaling pathways associated with cell proliferation and cell viability. The relevance of Hsp90 as a therapeutic target for numerous diseases states has prompted the identification and optimization of novel Hsp90 inhibitors as an emerging therapeutic strategy. We performed a screening aimed to identify novel Hsp90 inhibitors among several natural compounds and we focused on the iminosugar (+)-lentiginosine, a natural amyloglucosidases inhibitor, for its peculiar bioactivity profile. Characterization of Hsp90 inhibition was performed using a panel of chemical and biological approaches, including limited proteolysis, biochemical and cellular assays. Our result suggested that the middle domain of Hsp90, as opposed to its ATP-binding pocket, is a promising binding site for new classes of Hsp90 inhibitors with multitarget anti-cancer potentia

A Chemical-Biological Study Reveals C-9-type Iridoids as Novel Heat Shock Protein 90 (Hsp90) Inhibitors

The potential of heat shock protein 90 (Hsp90) as a therapeutic target for numerous diseases has made the identification and optimization of novel Hsp90 inhibitors an emerging therapeutic strategy. A surface plasmon resonance (SPR) approach was adopted to screen some iridoids for their Hsp90 alpha binding capability. Twenty-four iridoid derivatives, including 13 new natural compounds, were isolated from the leaves of Tabebuia argentea and petioles of Catalpa bignonioides. Their structures were elucidated by NMR, electrospray ionization mass spectrometry, and chemical methods. By means of a panel of chemical and biological approaches, four iridoids were demonstrated to bind Hsp90 alpha. In particular, the dimeric iridoid argenteoside A was shown to efficiently inhibit the chaperone in biochemical and cellular assays. Our results disclose C-9-type iridoids as a novel class of Hsp90 inhibitors

Carnosol Attenuates LPS-Induced Inflammation of Cardiomyoblasts by Inhibiting NF-κB: A Mechanistic in Vitro and in Silico Study

Carnosol possesses several beneficial pharmacological properties. However, its role in lipopolysaccharide (LPS) induced inflammation and cardiomyocyte cell line (H9C2) has never been investigated. Therefore, the effect of carnosol and an NF-kappa B inhibitor BAY 11-7082 was examined, and the underlying role of the NF-kappa B-dependent inflammatory pathway was analyzed as the target enzyme. Cell viability, inflammatory cytokines levels (tumor necrosis factor-alpha (TNF-alpha), interleukin (IL)-1 beta, IL-6, and prostaglandin E-2 (PGE(2))), and related gene expression (TNF-alpha, IL-1 beta, IL-6, and cyclooxygenase-2 (COX-2)) were analyzed by ELISA and real-time PCR. In addition, docking studies analyzed carnosol's molecular interactions and binding modes to NF-kappa B and IKK. We report that LPS caused the reduction of cell viability while enhancing both cytokines protein and mRNA levels (P &lt; 0.001, for all cases). However, the BAY 11-7082 pretreatment of the cells and carnosol increased cell viability and reduced cytokine protein and mRNA levels (P &lt; 0.001 vs. LPS, for all cases). Furthermore, our in silico analyses also supported the modulation of NF-kappa B and IKK by carnosol. This evidence highlights the defensive effects of carnosol against sepsis-induced myocardial dysfunction and, contextually, paved the rationale for the next in vitro and in vivo studies aimed to precisely describe its mechanism(s) of action

Breed and Feeding System Impact the Bioactive Anti-Inflammatory Properties of Bovine Milk

In the present study, we aimed at assessing the influence of breed and feeding system on the bovine milk profile of betaines and carnitines and milk capacity in counteracting the inflammatory endothelial cell (EC) damage induced by interleukin (IL)-6. In the first experimental design, two breeds were chosen (Holstein vs. Modicana) to investigate the biomolecule content and antioxidant capacity in milk and dairy products. In the second experimental design, two feeding systems (pasture vs. total mixed ratio) were tested only in Holstein to evaluate the possible effect on the functional profile of milk and dairy products. Finally, the bulk milk from the two experimental designs was used to evaluate the efficacy of preventing IL-6-induced endothelial inflammatory damage. Results showed that Modicana milk and whey had higher biomolecule content and antioxidant activity compared to Holstein milk (p < 0.01). Milk from Holstein fed TMR showed higher concentration of γ-butyrobetaine, δ-valerobetaine (p < 0.01), and l-carnitine (p < 0.05). Similarly, whey from Holstein fed TMR also showed higher content of δ-valerobetaine, glycine betaine, l-carnitine, and acetyl-l-carnitine (p < 0.01) compared to the Holstein fed pasture. Conversely, the antioxidant activity of milk and dairy products was not affected by the feeding system. In ECs, all milk samples reduced the IL-6-induced cytokine release, as well as the accumulation of reactive oxygen species (ROS) and the induction of cell death, with the most robust effect elicited by Modicana milk (p < 0.01). Overall, Modicana milk showed a higher content of biomolecules and antioxidant activity compared to Holstein, suggesting that the breed, more than the feeding system, can positively affect the health-promoting profile of dairy cattle milk

Exploring the Anticancer Potential of Premna resinosa (Hochst.) Leaf Surface Extract: Discovering New Diterpenes as Heat Shock Protein 70 (Hsp70) Binding Agents

Premna, a genus consisting of approximately 200 species, predominantly thrives in tropical and subtropical areas. Many of these species have been utilized in ethnopharmacology for diverse medicinal applications. In Saudi Arabia, Premna resinosa (Hochst.) Schauer (Lamiaceae) grows wildly, and its slightly viscid leaves are attributed to the production of leaf accession. In this study, we aimed to extract the surface accession from fresh leaves using dichloromethane to evaluate the anticancer potential. The plant exudate yielded two previously unknown labdane diterpenes, Premnaresone A and B, in addition to three already described congeners and four known flavonoids. The isolation process was accomplished using a combination of silica gel column chromatography and semi preparative HPLC, the structures of which were identified by NMR and HRESIMS analyses and a comparison with the literature data of associated compounds. Furthermore, we employed a density functional theory (DFT)/NMR approach to suggest the relative configuration of different compounds. Consequently, we investigated the possibility of developing new chaperone inhibitors by subjecting diterpenes 1–5 to a Surface Plasmon Resonance-screening, based on the knowledge that oridonin, a diterpene, interacts with Heat Shock Protein 70 (Hsp70) 1A in cancer cells. Additionally, we studied the anti-proliferative activity of compounds 1–5 on human Jurkat (human T-cell lymphoma) and HeLa (epithelial carcinoma) cell lines, where diterpene 3 exhibited activity in Jurkat cell lines after 48 h, with an IC50 of 15.21 ± 1.0 µM. Molecular docking and dynamic simulations revealed a robust interaction between compound 3 and Hsp70 key residues