Influence of energy supplementation on dietary nitrogen utilization and milk production in cows fed foliage of Leucaena leucocephala.

The aim of the study was to assess the effect of four energy supplements (two highly fermentable; two starch-based carbohydrates) on blood urea nitrogen (BUN), urinary urea excretion, and milk yield, in dual-purpose cows fed foliage of Leucaena leucocephala (Leucaena). Five Holstein-Zebu cows with 450 kg body weight in their second third of lactation were used in a 5 × 5 Latin square design. Cows were fed (dry basis) a mixture of 45% Leucaena and 55% Pennisetum purpureum grass. Treatments were supplementation with (i) sugarcane molasses (Mo), (ii) sorghum grain (So), (iii) fresh citrus pulp (CitP) or (iv) rice polishing (RP), all of them incorporated into the diet at 25 MJ of ME/cow/day. There was a control group (Cont) without energy supplementation. The study comprised five periods of 20 days (15 days adaptation, 5 days measurements). Dry matter intake (kg/day) was lower (P < 0.05) for Cont (9.4) compared with Mo (12.1), So (12.0), CitP (11.9) and RP (11.9) but no difference was observed among energy supplements (P > 0.05). Milk yield (kg/day) was higher (P < 0.05) in cows supplemented with starch supplements (4.7 for So; 4.9 for RP) compared with Cont (3.3). Milk yield from highly fermentable supplements (Mo and CitP) did not differ (P > 0.05) from Cont or other treatments. Milk protein, fat and lactose were not different among treatments (P > 0.05). Blood urea nitrogen and urinary urea excretion were both reduced (P < 0.05) by energy supplementation. Urinary urea excretion was not different (P > 0.05) among cows fed different sources of energy. It is concluded that in dual-purpose cows fed Leucaena foliage, supplementation with sugarcane molasses, citrus pulp or rice polishing reduced blood urea nitrogen and urinary urea excretion. Milk yield was increased by sorghum and rice polishing whereas energy supplementation did not affect milk composition.

Rumen function in vivo and in vitro in sheep fed Leucaena leucocephala.

The effect of Leucaena leucocephala inclusion in sheep diets upon rumen function was evaluated. Nine Pelibuey sheep, 32.6 ± 5.33 kg live weight (LW), fitted with rumen cannula were used. A complete randomized block design was employed. Two experimental periods of 60 days each, with 60-day intervals between them, were used. Experimental treatments were as follows (n = 6): T1 (control), 100 % Pennisetum purpureum grass; T2, 20 % L. leucocephala + 80 % P. purpureum; T3, 40 % L. leucocephala + 60 % P. purpureum. In situ rumen neutral detergent fiber (aNDF) and crude protein (CP) degradation, dry matter intake (DMI), volatile fatty acids (VFA) production, estimated methane (CH4) yield, rumen pH, ammonia nitrogen (N-NH3), and protozoa counts were measured. The aNDF in situ rumen degradation of P. purpureum and leucaena was higher (P < 0.05) in T2 and T3. Leucaena CP degradation was higher in T2 and T3 but for P. purpureum it was only significantly higher in T3. Leucaena aNDF and CP degradation rate (c) was 50 % higher (P < 0.05) in T2 and T3, but only higher in T3 for P. purpureum. Voluntary intake and rumen (N-NH3) was higher in T2 and T3 (P = 0.0001, P = 0.005, respectively). Molar VFA proportions were similar for all treatments (P > 0.05). Protozoa counts and in vitro gas production (48 h) were lower in T2 and T3 (P < 0.05, P < 0.0001). Estimated methane yield (mol CH4/day) was higher in sheep fed leucaena (P < 0.0001). However, CH4 yield relative to animal performance (mol CH4/g LW gain) was lower in T2 and T3 (P < 0.0001). In summary, these results indicate that including L. leucocephala in sheep diets did not modify rumen fermentation pattern (same VFA ratios) nor reduce the amount of CH4 per unit of DMI (mol CH4/g DMI). However, leucaena inclusion does increase rumen N-NH3, aNDF and CP digestibility, and voluntary intake.

Sequential combination of decitabine and idarubicin synergistically enhances anti-leukemia effect followed by demethylating Wnt pathway inhibitor promoters and downregulating Wnt pathway nuclear target.

BACKGROUND: The methylation inhibitor 5-Aza-2'-deoxycytidine (decitabine, DAC) has a great therapeutic value for acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS). But decitabine monotherapy was associated with a relatively low rate of complete remission in AML and MDS. We aimed to investigate the effect of several anti-leukemia drugs in combination with decitabine on the proliferation of myeloid leukemia cells, to select the most efficient combination group and explore the associated mechanisms of these combination therapies. METHODS: Cell proliferation was tested by MTT assay and CFU-GM assay. Cell apoptosis was evaluated by Annexin V and PI staining in cell culture, TUNEL assay and transmission electron microscopy in animal study. MicroPET was used to imaging the tumor in mouse model. Molecular studies were conducted using microarray expression analysis, which was used to explore associated pathways, and real-time quantitative reverse transcription-PCR, western blot and immunohistochemistry, used to assess regulation of Wnt/ß-catenin pathway. Statistical significance among groups was determined by one-way ANOVA analysis followed by post hoc Bonferroni's multiple comparison test. RESULTS: Among five anti-leukemia agents in combining with decitabine, the sequential combination of decitabine and idarubicin induced synergistic cell death in U937 cells, and this effect was verified in HEL, SKM-1 cells and AML cells isolated from AML patients. Importantly, tumor growth inhibition in this sequential combination was found to be higher than in single agent or controls in vivo. Moreover, sequential combination of the two agents induced apoptosis and depression of the Wnt/ß-catenin pathway in both AML cell culture and animal studies. CONCLUSIONS: The findings demonstrated that sequentially combination of decitabine and idarubicin had synergistic anti-leukemia effects. These effects were mainly attributed to demethylation of Wnt/ß-catenin pathway inhibitors and downregulation of Wnt/ß-catenin pathway nuclear targets.

Effect of Brosimum alicastrum Foliage on Intake, Kinetics of Fermentation and Passage and Microbial N Supply in Sheep Fed Megathyrsus maximus Hay.

An experiment was carried out to assess the effect of the incorporation of sun-dried foliage of Brosimum alicastrum into rations based on hay of Megathyrsus maximus on intake, rumen fermentation, kinetics of passage, microbial nitrogen supply to the small intestine, apparent digestibility in Pelibuey hair sheep. Four rations were randomly allotted to four rumen-cannulated lambs (BW = 37.4 ± 4.9 kg) using a 4 × 4 Latin square design to assess the effect of increasing levels (0, 15, 30 and 45% DM basis) of foliage of Brosimum alicastrum on a basal ration of M. maximus. Organic matter intake and water consumption increased linearly (p < 0.01) with increasing levels of B. alicastrum in the ration. The rate and potential extent of rumen fermentation of OM and CP of B. alicastrum were 10.6%/h and 86.6% and 11.4%/h and 95.2%, respectively, but no effect (p > 0.05) was found on the potential rumen degradation of DM (40.2%) or on the rate of degradation of DM (0.033%/h) of M. maximus, although a positive effect was found in the rumen degradation rate of NDF (p < 0.05). VFA and ammonia concentration in the rumen and the rate of passage of solids and liquids through the rumen (k1) increased linearly (p < 0.01) with increasing levels of B. alicastrum. Rumen pH was not affected by the incorporation of B. alicastrum (p > 0.05). Microbial nitrogen supply to the small intestine (p < 0.001), apparent digestibility of dry matter (p < 0.01) and NDF (p < 0.05) of the rations were also significantly increased as a result of the incorporation of B. alicastrum foliage. Results from this experiment suggest that the foliage of Brosimum alicastrum can be readily incorporated at around 30% of the ration of dry matter in hair sheep with beneficial effects on feed intake, rate of passage and microbial N supply to the lower tract.

Dry matter intake and digestibility of rations replacing concentrates with graded levels of Enterolobium cyclocarpum in Pelibuey lambs.

The aim of the study was to evaluate the effect of graded levels of Enterolobium cyclocarpum pods in the ration on feed intake and digestibility by Pelibuey lambs. Five dietary treatments were imposed where ground pods replaced concentrate diet at 0, 20, 30, 40 and 50 % of dry matter (DM), respectively. The concentrate portion was composed of ground sorghum, soybean meal, cane molasses and minerals. Five entire Pelibuey lambs with initial bodyweight 34 ± 2 kg were allocated in the treatments in a 5 × 5 Latin square design. Values of dry matter intake (DMI) and dry matter (DMD) and organic matter (OMD) digestibility were measured and metabolisable energy intake (MEI) estimated. Rumen degradation constants for E. cyclocarpum were also measured. There were no differences (P > 0.05) in average DMI (86.6 g/kg(0.75)) and OMI (81.2 g/kg(0.75)) among treatments. As the level of incorporation of E. cyclocarpum pods increased, voluntary DMI and OMI increased, whereas apparent DMD and OMD decreased linearly. Average digestible DM (65 g/kg(0.75)) and OM (61 g/kg(0.75)) intakes were similar (P > 0.05) among treatments. Similarly, MEI (0.976 MJ ME kg(0.75)/day) was not different (P > 0.05) among treatments. The potential rumen degradation (A + B) of ground pods of E. cyclocarpum was 866.4 g/kg DM. Ground pods of E. cyclocarpum can be employed for lamb feeding up to 50 % of the ration, without affecting DMI, DM apparent digestibility and MEI.

Pain Relief After Allogenic Stem Cell Disc Therapy.

BACKGROUND: Treatment of intermediate-stage painful degenerative disc disease is controversial, with few reliable options. Allogenic mesenchymal stem cells (MSCs)are an alternative to autologous stem cell transplantation. Allogeneic MSCs in the treatment of discogenic low back pain have some practical advantages, ranging from availability to ease of treatment in a procedure-room setting. OBJECTIVES: To assess the efficacy and safety of allogenic MSC injection into painful lumbar intervertebral discs and associated clinical outcomes. STUDY DESIGN: Retrospective observational cohort study. SETTING: Private practice. METHODS: There were 33 patients: 15 women and 18 men with an average age of 47.6 years. The patients' average follow-up was 26.88 months Patients were treated with intradiscal injection of approximately 5 million allogeneic polyclonal MSCs in 1% hyaluronic acid derived from immunoselected umbilical cord stem cells. Patients were monitored for adverse event reactions. Clinical outcomes were assessed with reductions in the reported Visual Analog Scale (VAS) for back pain, the Oswestry Disability Index (ODI) scores, and the use of the modified Macnab criteria. RESULTS: No patient required any additional treatments for low back pain stemming from the level treated with MSC injections. At a 2-year follow-up, the average VAS low back score reduction was 6.565 ± 1.619 and 38.333 ± 14.865 for the ODI (P < 0.001). Reported Macnab outcomes were excellent in 11 patients (33.3%), good in 19 (57.6%), and fair in 3 (9.1%). LIMITATIONS: Our observational study is limited by patient selection, hindsight bias, and low patient numbers. CONCLUSION: The results of our feasibility study suggest that the injection of allogeneic MSCs to treat patients with painful intermediate-stage degenerative disc disease has merit. No adverse reactions were observed. The authors recommend further study in a randomized prospective study setting with a placebo control group or a natural history study group of patients to solidify this research.

Case report: Neuronal intranuclear inclusion disease presenting with acute encephalopathy.

Neuronal intranuclear inclusion disease (NIID), a neurodegenerative disease previously thought to be rare, is increasingly recognized despite heterogeneous clinical presentations. NIID is pathologically characterized by ubiquitin and p-62 positive intranuclear eosinophilic inclusions that affect multiple organ systems, including the brain, skin, and other tissues. Although the diagnosis of NIID is challenging due to phenotypic heterogeneity, a greater understanding of the clinical and imaging presentations can improve accurate and early diagnosis. Here, we present three cases of pathologically proven adult-onset NIID, all presenting with episodes of acute encephalopathy with protracted workups and lengthy time between symptom onset and diagnosis. Case 1 highlights challenges in the diagnosis of NIID when MRI does not reveal classic abnormalities and provides a striking example of hyperperfusion in the setting of acute encephalopathy, as well as unique pathology with neuronal central chromatolysis, which has not been previously described. Case 2 highlights the progression of MRI findings associated with multiple NIID-related encephalopathic episodes over an extended time period, as well as the utility of skin biopsy for antemortem diagnosis.

Noncompetitive blocking of human GLUT1 hexose transporter by methylxanthines reveals an exofacial regulatory binding site.

Glucose transporter (GLUT)1 has become an attractive target to block glucose uptake in malignant cells since most cancer cells overexpress GLUT1 and are sensitive to glucose deprivation. Methylxanthines are natural compounds that inhibit glucose uptake; however, the mechanism of inhibition remains unknown. Here, we used a combination of binding and glucose transport kinetic assays to analyze in detail the effects of caffeine, pentoxifylline, and theophylline on hexose transport in human erythrocytes. The displacement of previously bound cytochalasin B revealed a direct interaction between the methylxanthines and GLUT1. Methylxanthines behave as noncompetitive blockers (inhibition constant values of 2-3 mM) in exchange and zero-trans efflux assays, whereas mixed inhibition with a notable uncompetitive component is observed in zero-trans influx assays (inhibition constant values of 5-12 mM). These results indicate that methylxanthines do not bind to either exofacial or endofacial d-glucose-binding sites but instead interact at a different site accessible by the external face of the transporter. Additionally, infinite-cis exit assays (Sen-Widdas assays) showed that only pentoxifylline disturbed d-glucose for binding to the exofacial substrate site. Interestingly, coinhibition assays showed that methylxanthines bind to a common site on the transporter. We concluded that there is a methylxanthine regulatory site on the external surface of the transporter, which is close but distinguishable from the d-glucose external site. Therefore, the methylxanthine moiety may become an attractive framework for the design of novel specific noncompetitive facilitative GLUT inhibitors.

Effects of energy supplementation on productivity of dual-purpose cows grazing in a silvopastoral system in the tropics.

The aim of the present work was to evaluate milk yield, postpartum (pp) ovarian activity and pregnancy rate in dual-purpose cows grazing Cynodon nlemfuensis and browsing L. leucocephala, with or without energy supplementation. Twenty-four Bos taurus × B. indicus cows were divided in two groups from calving to 70 days post-calving: supplemented group (SG) with ground sorghum grain offered at 0.4% of live weight at calving and control group (CG) without supplement. There was a trend for milk yield (kg day(-1)) to be greater (p = 0.08) for SG (10.55 ± 0.51) compared to CG (9.53 ± 0.61), although without differences in fat (0.42 ± 0.02 vs. 0.38 ± 0.03 kg day(-1)), protein (0.29 ± 0.02 vs. 0.29 ± 0.02 kg day(-1)) or lactose (0.49 ± 0.02 vs. 0.49 ± 0.03 kg day(-1)) concentration. Populations of large, medium and small follicles were similar between treatments. Percentage of cows which showed corpus luteum tended to be greater in SG (50%), compared to CG (33%). Supplemented cows tended to have a shorter calving-first corpus luteum interval (40 ± 10 vs. 51 ± 10 days) and had a significantly higher (χ (2) = 0.03) pregnancy rate (42% vs. 0%). It is concluded that energy supplementation helped to improve ovarian activity and pregnancy rate. Since supplementation did not avoid loss of body condition, the higher pregnancy rate in SG suggests beneficial effects of supplementation probably mediated by metabolic hormones.

Histidine residues in the Na+-coupled ascorbic acid transporter-2 (SVCT2) are central regulators of SVCT2 function, modulating pH sensitivity, transporter kinetics, Na+ cooperativity, conformational stability, and subcellular localization.

Na(+)-coupled ascorbic acid transporter-2 (SVCT2) activity is impaired at acid pH, but little is known about the molecular determinants that define the transporter pH sensitivity. SVCT2 contains six histidine residues in its primary sequence, three of which are exofacial in the transporter secondary structure model. We used site-directed mutagenesis and treatment with diethylpyrocarbonate to identify histidine residues responsible for SVCT2 pH sensitivity. We conclude that five histidine residues, His(109), His(203), His(206), His(269), and His(413), are central regulators of SVCT2 function, participating to different degrees in modulating pH sensitivity, transporter kinetics, Na(+) cooperativity, conformational stability, and subcellular localization. Our results are compatible with a model in which (i) a single exofacial histidine residue, His(413), localized in the exofacial loop IV that connects transmembrane helices VII-VIII defines the pH sensitivity of SVCT2 through a mechanism involving a marked attenuation of the activation by Na(+) and loss of Na(+) cooperativity, which leads to a decreased V(max) without altering the transport K(m); (ii) exofacial histidine residues His(203), His(206), and His(413) may be involved in maintaining a functional interaction between exofacial loops II and IV and influence the general folding of the transporter; (iii) histidines 203, 206, 269, and 413 affect the transporter kinetics by modulating the apparent transport K(m); and (iv) histidine 109, localized at the center of transmembrane helix I, might be fundamental for the interaction of SVCT2 with the transported substrate ascorbic acid. Thus, histidine residues are central regulators of SVCT2 function.

In utero transplantation of adult bone marrow decreases perinatal lethality and rescues the bone phenotype in the knockin murine model for classical, dominant osteogenesis imperfecta.

Autosomal dominant osteogenesis imperfecta (OI) caused by glycine substitutions in type I collagen is a paradigmatic disorder for stem cell therapy. Bone marrow transplantation in OI children has produced a low engraftment rate, but surprisingly encouraging symptomatic improvements. In utero transplantation (IUT) may hold even more promise. However, systematic studies of both methods have so far been limited to a recessive mouse model. In this study, we evaluated intrauterine transplantation of adult bone marrow into heterozygous BrtlIV mice. Brtl is a knockin mouse with a classical glycine substitution in type I collagen [alpha1(I)-Gly349Cys], dominant trait transmission, and a phenotype resembling moderately severe and lethal OI. Adult bone marrow donor cells from enhanced green fluorescent protein (eGFP) transgenic mice engrafted in hematopoietic and nonhematopoietic tissues differentiated to trabecular and cortical bone cells and synthesized up to 20% of all type I collagen in the host bone. The transplantation eliminated the perinatal lethality of heterozygous BrtlIV mice. At 2 months of age, femora of treated Brtl mice had significant improvement in geometric parameters (P < .05) versus untreated Brtl mice, and their mechanical properties attained wild-type values. Our results suggest that the engrafted cells form bone with higher efficiency than the endogenous cells, supporting IUT as a promising approach for the treatment of genetic bone diseases.

Energetic efficiency of milk synthesis in dual-purpose cows grazing tropical pastures.

The aim of this study was to assess the energetic efficiency of milk synthesis by grazing dual-purpose cows with or without a starch-based supplement in tropical South Mexico. Forty-six Holstein × Zebu cows were used in a 2 × 2 × 2 factorial design. Factors analysed were diet (supplemented, unsupplemented), age (young: 1-2 calvings, mature: >3 calvings) and day of lactation (21 and 84 days post-calving). The supplement represented about 30% of estimated dry matter (DM) intake. Grass intake was measured using the n-alkane technique at 21 and 84 days post-calving when calculations of efficiency were performed. Efficiency for milk synthesis was reported as feed conversion efficiency (FCE, kilograms of milk per kilogram of DM intake), gross energetic efficiency (GEE, milk energy output/metabolisable energy (ME) intake) and efficiency of ME use for lactation (k(l), adjusted to zero energy balance). There were no interactions between factors. FCE and GEE were not different between diets, but supplemented cows had a lower (p < 0.01) k(l) value (0.62) than unsupplemented cows (0.67), suggesting a diverted partition of nutrients towards body tissue. Mature cows were more efficient (p < 0.001) than young cows in terms of FCE (1.13 vs 0.87) and GEE (0.34 vs 0.26), but equal in terms of k(l) (0.65). FCE (1.10 vs 0.90) and GEE (0.34 vs 0.27) were both higher on day 21 compared with day 84 post-calving, with a trend for a higher k(l) in early lactation. Dual-purpose cows used tropical grasses efficiently for milk synthesis, and higher milk yield observed in supplemented cows was due to a higher intake of nutrients rather than a higher energetic efficiency.

Effect of Chitosan and Naringin on Enteric Methane Emissions in Crossbred Heifers Fed Tropical Grass.

In order to meet consumer needs, the livestock industry is increasingly seeking natural feed additives with the ability to improve the efficiency of nutrient utilization, alternatives to antibiotics, and mitigate methane emissions in ruminants. Chitosan (CHI) is a polysaccharide with antimicrobial capability against protozoa and Gram-positive and -negative bacteria, fungi, and yeasts while naringin (NA) is a flavonoid with antimicrobial and antioxidant properties. First, an in vitro gas production experiment was performed adding 0, 1.5, 3.0 g/kg of CHI and NA under a completely randomized design. The substrate containing forage and concentrate in a 70:30 ratio on a dry matter (DM) basis. Compounds increased the concentration of propionic acid, and a significant reduction in methane production was observed with the inclusion of CHI at 1.5 g/kg in in vitro experiments (p < 0.001). In a dry matter rumen degradability study for 96 h, there were no differences in potential and effective degradability. In the in vivo study, six crossbred heifers fitted with rumen cannulas were assigned to a 6 × 6 Latin square design according to the following treatments: control (CTL), no additive; chitosan (CHI1, 1.5 g/kg DMI); (CHI2, 3.0 g/kg DMI); naringin (NA1, 1.5 g/kg DMI); (NA2, 3.0 g/kg DMI) and a mixture of CHI and NA (1.5 + 1.5 g/kg DMI) given directly through the rumen cannula. Additives did not affect rumen fermentation (p > 0.05), DM intake and digestibility of (p > 0.05), and enteric methane emissions (p > 0.05). CHI at a concentration of 1.5 g/kg DM in in vitro experiments had a positive effect on fermentation pattern increasing propionate and reduced methane production. In contrast, in the in vivo studies, there was not a positive effect on rumen fermentation, nor in enteric methane production in crossbred heifers fed a basal ration of tropical grass.

Construction and Operation of a Respiration Chamber of the Head-Box Type for Methane Measurement from Cattle.

This paper aims to describe the construction and operation of a respiration chamber of the head-box type for methane (CH4) measurements in bovines. The system consists of (1) a head box with a stainless steel frame and acrylic walls, floor, and ceiling; (2) a stainless steel feeder; (3) an automatic drinking water bowl; (4) a hood made from reinforced canvas; (5) an infrared (IR) CH4 gas analyzer, a mass flow generator, a data-acquisition system; and (6) a steel metabolic box. Six assays were conducted to determine the pure CH4 recovery rate of the whole system in order to validate it and comply with standards of chamber operation. The gravimetrical method was used for the recovery test and the recovery rate obtained was 1.04 ± 0.05. Once the system was calibrated, measurements of CH4 were conducted using eight animals consisting of four Holstein cows with a live weight of 593.8 ± 51 kg and an average milk yield of 23.3 ± 1.8 kg d-1 and four heifers with a live weight of 339 ± 28 kg. The CH4 production values were 687 ± 123 and 248 ± 40 L CH4 d-1 for cows and heifers, respectively. The CH4 yield was 19.7 ± 3.4 g and 17.1 ± 3.4 g CH4 kg-1 of dry matter consumed for cows and heifers, respectively. These results are consistent with those reported in the literature.

Role of Secondary Plant Metabolites on Enteric Methane Mitigation in Ruminants.

The rumen microbiome plays a fundamental role in all ruminant species, it is involved in health, nutrient utilization, detoxification, and methane emissions. Methane is a greenhouse gas which is eructated in large volumes by ruminants grazing extensive grasslands in the tropical regions of the world. Enteric methane is the largest contributor to the emissions of greenhouse gases originating from animal agriculture. A large variety of plants containing secondary metabolites [essential oils (terpenoids), tannins, saponins, and flavonoids] have been evaluated as cattle feedstuffs and changes in volatile fatty acid proportions and methane synthesis in the rumen have been assessed. Alterations to the rumen microbiome may lead to changes in diversity, composition, and structure of the methanogen community. Legumes containing condensed tannins such as Leucaena leucocephala have shown a good methane mitigating effect when fed at levels of up to 30-35% of ration dry matter in cattle as a result of the effect of condensed tannins on rumen bacteria and methanogens. It has been shown that saponins disrupt the membrane of rumen protozoa, thus decreasing the numbers of both protozoa and methanogenic archaea. Trials carried out with cattle housed in respiration chambers have demonstrated the enteric methane mitigation effect in cattle and sheep of tropical legumes such as Enterolobium cyclocarpum and Samanea saman which contain saponins. Essential oils are volatile constituents of terpenoid or non-terpenoid origin which impair energy metabolism of archaea and have shown reductions of up to 26% in enteric methane emissions in ruminants. There is emerging evidence showing the potential of flavonoids as methane mitigating compounds, but more work is required in vivo to confirm preliminary findings. From the information hereby presented, it is clear that plant secondary metabolites can be a rational approach to modulate the rumen microbiome and modify its function, some species of rumen microbes improve protein and fiber degradation and reduce feed energy loss as methane in ruminants fed tropical plant species.

Effect of Dried Leaves of Leucaena leucocephala on Rumen Fermentation, Rumen Microbial Population, and Enteric Methane Production in Crossbred Heifers.

The effects of dietary inclusion of dried Leucaena leucocephala leaves (DLL) on nutrient digestibility, fermentation parameters, microbial rumen population, and production of enteric methane (CH4) in crossbred heifers were evaluated. Four heifers were used in a 4 × 4 Latin square design consisting of four periods and four levels of inclusion of DLL: 0%, 12%, 24%, and 36% of dry matter (DM) intake. Results showed that DM intake (DMI), organic matter intake, and gross energy intake (GEI) were similar (p > 0.05) among treatments. Apparent digestibility of organic matter, neutral detergent fiber, and energy decreased with increasing levels of DLL in the ration (p < 0.05). In contrast, digestible crude protein (CP) was higher (p < 0.05) in treatments with 12% and 24% DM of DLL. The inclusion of DLL did not affect (p > 0.05) rumen pH and total volatile fatty acids. Rumen microbial community was not affected (p > 0.05) by treatment. There was a linear reduction (p < 0.05) in CH4 emissions as the levels of DLL in the ration were increased. Results of this study suggest that an inclusion of 12% DM of ration as DLL enhances digestible CP and reduces daily production of enteric CH4 without adversely affecting DMI, rumen microbial population, and fermentation parameters.

GLUT1 and GLUT8 support lactose synthesis in Golgi of murine mammary epithelial cells.

The mammary gland increases energy requirements during pregnancy and lactation to support epithelial proliferation and milk nutrients synthesis. Lactose, the principal carbohydrate of the milk, is synthetized in the Golgi of mammary epithelial cells by lactose synthase from glucose and UPD galactose. We studied the temporal changes in the expression of GLUT1 and GLUT8 in mammary gland and their association with lactose synthesis and proliferation in BALB/c mice. Six groups were used: virgin, pregnant at 2 and 17 days, lactating at 2 and 10 days, and weaning at 2 days. Temporal expression of GLUT1 and GLUT8 transporters by qPCR, western blot and immunohistochemistry, and its association with lactalbumin, Ki67, and cytokeratin 18 within mammary tissue was studied, along with subcellular localization. GLUT1 and GLUT8 transporters increased their expression during mammary gland progression, reaching 20-fold increasing in GLUT1 mRNA at lactation (p < 0.05) and 2-fold at protein level for GLUT1 and GLUT8 (p < 0.05 and 0.01, respectively). The temporal expression pattern was shared with cytokeratin 18 and Ki67 (p < 0.01). Endogenous GLUT8 partially co-localized with 58 K protein and α-lactalbumin in mammary tissue and with Golgi membrane-associated protein 130 in isolated epithelial cells. The spatial-temporal synchrony between expression of GLUT8/GLUT1 and alveolar cell proliferation, and its localization in cis-Golgi associated to lactose synthase complex, suggest that both transporters are involved in glucose uptake into this organelle, supporting lactose synthesis.

The Role of Chitosan as a Possible Agent for Enteric Methane Mitigation in Ruminants.

Livestock production is a main source of anthropogenic greenhouse gases (GHG). The main gases are CH4 with a global warming potential (GWP) 25 times and nitrous oxide (N2O) with a GWP 298 times, that of carbon dioxide (CO2) arising from enteric fermentation or from manure management, respectively. In fact, CH4 is the second most important GHG emitted globally. This current scenario has increased the concerns about global warming and encouraged the development of intensive research on different natural compounds to be used as feed additives in ruminant rations and modify the rumen ecosystem, fermentation pattern, and mitigate enteric CH4. The compounds most studied are the secondary metabolites of plants, which include a vast array of chemical substances like polyphenols and saponins that are present in plant tissues of different species, but the results are not consistent, and the extraction cost has constrained their utilization in practical animal feeding. Other new compounds of interest include polysaccharide biopolymers such as chitosan, mainly obtained as a marine co-product. As with other compounds, the effect of chitosan on the rumen microbial population depends on the source, purity, dose, process of extraction, and storage. In addition, it is important to identify compounds without adverse effects on rumen fermentation. The present review is aimed at providing information about chitosan for dietary manipulation to be considered for future studies to mitigate enteric methane and reduce the environmental impact of GHGs arising from livestock production systems. Chitosan is a promising agent with methane mitigating effects, but further research is required with in vivo models to establish effective daily doses without any detrimental effect to the animal and consider its addition in practical rations as well as the economic cost of methane mitigation.

Effects of long-term diet supplementation with Gliricidia sepium foliage mixed with Enterolobium cyclocarpum pods on enteric methane, apparent digestibility, and rumen microbial population in crossbred heifers1.

In the last decades, strategies have been evaluated to reduce rumen methane (CH4) production by supplementing tropical forages rich in secondary compounds; however, most of these beneficial effects need to be validated in terms of their persistence over time. The aim of this study was to assess CH4 emissions over time in heifers fed with and without Gliricidia sepium foliage (G) mixed with ground pods of Enterolobium cyclocarpum(E). Two groups of 4 crossbred (Bos taurus x Bos indicus) heifers (284 ±17 kg initial weight) were fed with 2 diets (0% and 15% of a mixture of the pods and foliage [E + G:0 and E + G:15, respectively]) over 80 d, plus 2 wk before the experiment, in which every animal was fed a legume and pod-free diet. Every 14 d, CH4 production, apparent digestibility, volatile fatty acids (VFA), and microbial population were quantified for each animal. The experiment was conducted with a repeated measurements design over time. Diets fed differed in terms of their crude protein (CP), condensed tannins, and saponins content supplied by E. cyclocarpum and G. sepium. For most of the experiment, dry matter intake (DMI) and digestible dry-matter intake (DDMI) were 6.3 kg DMI/d and 512 g DDMI/kg, respectively, for both diets (diet: P > 0.05). Apparent digestible crude protein (DCP) was reduced by 21 g DCP/kg DM when the diet was supplemented with E + G:15 (P = 0.040). Molar proportions of VFA's in the rumen did not differ between diets or in time (P > 0.05). Daily methane production, expressed in relation to DMI, was 23.95 vs. 23.32 g CH4/kg DMI for the diet E + G:0 and E + G:15, respectively (diet: P = 0.016; Time: P > 0.05). Percent gross energy loss as CH4 (Ym) with grass-only diets was above 8.1%, whereas when feeding heifers with the alternate supplementation, Ym values of 7.59% (P = 0.016) were observed. The relative abundance of total bacterial, protozoa, and methanogenic archaeal replicates was not affected by time nor by the incorporation of legume and pods into the diet (P > 0.05). Results suggest that addition of G. sepium mixed with E. cyclocarpum pods can reduce CH4 production in heifers and this response remains over time, without effect on microbial population and VFA concentration and a slight reduction in CPD digestibility.

Increased expression of mitochondrial sodium-coupled ascorbic acid transporter-2 (mitSVCT2) as a central feature in breast cancer.

The potential role of vitamin C in cancer prevention and treatment remains controversial. While normal human cells obtain vitamin C as ascorbic acid, the prevalent form of vitamin C in vivo, the uptake mechanisms by which cancer cells acquire vitamin C has remained unclear. The aim of this study is to characterize how breast cancer cells acquire vitamin C. For this, we determined the expression of vitamin C transporters in normal and breast cancer tissue samples, and in ZR-75, MCF-7, MDA-231 and MDA-468 breast cancer cell lines. At the same time, reduced (AA) and oxidized (DHA) forms of vitamin C uptake experiments were performed in all cell lines. We show here that human breast cancer tissues differentially express a form of SVCT2 transporter, that is systematically absent in normal breast tissues and it is increased in breast tumors. In fact, estrogen receptor negative breast cancer tissue, exhibit the most elevated SVCT2 expression levels. Despite this, our analysis in breast cancer cell lines showed that these cells are not able to uptake ascorbic acid and depend on glucose transporter for the acquisition of vitamin C by a bystander effect. This is consistent with our observations that this form of SVCT2 is completely absent from the plasma membrane and is overexpressed in mitochondria of breast cancer cells, where it mediates ascorbic acid transport. This work shows that breast cancer cells acquire vitamin C in its oxidized form and are capable of accumulated high concentrations of the reduced form. Augmented expression of an SVCT2 mitochondrial form appears to be a common hallmark across all human cancers and might have implications in cancer cells survival capacity against pro-oxidant environments.