Non-encapsulated, encapsulated, and lyophilized probiotic Limosilactobacillus reuteri SW23 influenced the growth and gut health in calves.

The present study was conducted to assess the impact of non-encapsulated, air-dried microencapsulated, and lyophilized microencapsulated probiotics in indigenous cattle calves (Bos indicus). Twenty-four (5-7 days old) indigenous cattle calves were selected and assigned into four groups, with six calves in each as follows: control (CON), fed milk and basal diet alone, and treatment groups supplemented with non-encapsulated (NEC), air-dried microencapsulated (AEC) and lyophilized microencapsulated (LEC) probiotic L. reuteri SW23 at 108 CFU/head/day in skim milk as a carrier provided for 60 days. The animals were divided into four groups, adopting a complete randomized design, and the effects were considered significant at p ≤ 0.05. Probiotics supplementation increased (p < 0.05) body weight gain (kg), average daily gain, and structural growth measurements in calves of all treatment groups. Dry matter intake (g/d), feed conversion efficiency, and fecal counts of Lactobacilli and Bifidobacteria were also increased in the treatment groups compared to CON. The fecal consistency index was highest in CON (0.70 ± 0.03), followed by NEC (0.68 ± 0.01), AEC (0.66 ± 0.02), and LEC (0.65 ± 0.02). Fecal pH and ammonia levels were reduced (p < 0.05) in the probiotic-fed groups compared to CON, with a concomitant increase in fecal lactate, acetate, and propionate levels. In addition, cell-mediated and humoral immunity were significantly increased in supplemented groups as compared to CON. Thus, it can be concluded that supplementation of the probiotics in microencapsulated/non-encapsulated forms to neonatal calves had a variety of positive effects on their health, including better performance, improved gut health, and a lower fecal consistency index. Moreover, among all supplemented groups, the lyophilized microencapsulated group outperformed air-dried microencapsulated and non-microencapsulated groups in terms of ADG, DMI, and gut health.

Impact of synbiotics on growth performance and gut health in Murrah buffalo calves.

Synbiotics have been used as biotherapeutic supplements for prevention of new-born calf gastrointestinal disorders. Present study was conducted to evaluate the impact of fructo-oligosaccharide, mannan-oligosaccharide and inulin along with Lactobacillus plantarum CRD-7 and Lactobacillus acidophilus NCDC15 on the nutrient digestibility, growth performance and faecal microbial population of pre-ruminant buffalo calves. Twenty-four Murrah calves (5 days old) were randomly assigned to four groups of six calves in each using randomized block design. Calves in Group I (control) received only a basic diet of milk, calf starter and berseem with no additives. Calves in Group II (SYN1) were fed 6 g fructo-oligosaccharide (FOS) + Lactobacillus plantarum CRD-7 (100 ml). Calves in Group III (SYN2) were fed 9 g inulin + L. plantarum CRD-7 (50 ml), while calves in Group IV (SYN3) received 4 g MOS + L. acidophilus NCDC15 (200 ml) as fermented milk having 108 CFU/ml/calf/day in addition to the basal diet. The results revealed that digestibility of dry matter, crude protein, ether extract and average daily gain were all higher (P < 0.05) in SYN1 as compared to control group. The antioxidant enzyme activity, humoral and cell mediated immunity performed well in SYN1, SYN2 and SYN3 as compared to control. Diarrhoea and faecal scouring were lower (P < 0.05) in all supplemented groups than control. Faecal Lactobacilli and Bifidobacterium counts were also higher in SYN1 group followed by SYN2 and SYN3. Faecal ammonia, lactate, pH, and volatile fatty acids level were increased in SYN1 supplemented groups. The synbiotic combination of 6 g FOS + L. plantarum CRD-7 had better response on digestibility, average daily gain, antioxidant enzymes, immune response, faecal microbiota and metabolites and also reduce the faecal score and diarrhoea incidence. Therefore, supplementation of 6 g FOS + L. plantarum CRD-7 can be advised for general use in order to promote long-term animal production.

Development of a Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS) Method for Characterizing Linalool Oral Pharmacokinetics in Humans.

Lavender (Lavandula angustifolia Miller or Lavandula officinalis Chaix) is an ethnopharmacological plant commonly known as English lavender. Linalool and linalyl acetate are putative phytoactives in lavender essential oil (LEO) derived from the flower heads. LEO has been used in aroma or massage therapy to reduce sleep disturbance and to mitigate anxiety. Recently, an oral LEO formulation was administered in human clinical trials designed to ascertain its anxiolytic effect. However, human pharmacokinetics and an LC-MS/MS method for the measurement of linalool are lacking. To address this deficiency, a rapid and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed for the analysis of linalool in human serum. Prior to the analysis, a simple sample preparation protocol including protein precipitation and liquid-liquid extraction of serum samples was created. The prepared samples were analyzed using a C18 reversed-phase column and gradient elution (acetonitrile and water, both containing 0.1% formic acid). A Waters Xevo TQ-S tandem mass spectrometer (positive mode) was used to quantitatively determine linalool and IS according to transitions of m/z 137.1→95.1 (tR 0.79 min) and 205.2→149.1 (tR 1.56 min), respectively. The method was validated for precision, accuracy, selectivity, linearity, sensitivity, matrix effects, and stability, and it was successfully applied to characterize the oral pharmacokinetics of linalool in humans. The newly developed LC-MS/MS-based method and its application in clinical trial serum samples are essential for the characterization of potential pharmacokinetic and pharmacodynamic interactions.

Comparative study of depotash vinasse and molasses as binder and production performance in lactating Murrah buffaloes.

Dairy sector has recently focused a lot of attention on the addition of agricultural by-products as functional feed additives as an environmentally friendly and sustainable technology. Depotash vinasse (DPV) serves as a cheap source of nutrients and a binder for animal feed in dairy sector. However, there is little information available on the usage of depotash vinasse on animals. Therefore, the aim of the present study was to assess the role of depotash vinasse as pellet binder on nutrient digestibility, blood parameters and milk production in early lactating Murrah buffaloes. Fifteen Murrah buffaloes (daily milk yield 8.5 to 9.0 kg/day) were randomly assigned to three groups, viz., control, group 1 (G1) and group 2 (G2) on the basis of milk yield and days in milk. The control group animals received a basal diet of concentrate mix, oat greens and wheat straw, G1 animals received molasses as a binder (8%), while G2 received DPV as binder (8%). Results revealed that there was no significant effect on nutrient digestibility. Blood parameters and hepatic enzymes were statistically similar (P > 0.05). Supplementation of depotash vinasse as binder had no effect on plasma minerals and was comparable to control group. There were no changes in milk production and 6% fat-corrected milk yield in treated groups as compared to control. It was concluded that depotash vinasse (8%) may be used for pellet production with no negative impact on milk yield and composition, nutrient digestibility and blood biochemical parameters in early lactating buffaloes.

Impact of zinc, copper, manganese and chromium supplementation on growth performance and blood metabolic profile of Sahiwal (Bos indicus) male calves.

This study aimed to see the effect of oral supplementation of specific trace minerals mixture on the growth, immunity, and reproductive development of indigenous growing bull calves. Eighteen Sahiwal bull calves, with an average age of 6 months were chosen and divided into three groups. Group 1 was fed with a basal diet, Group 2 was provided with an additional specific trace mineral supplement to achieve a diet containing 70 ppm of Zn, 17.50 ppm of Cu, 65 ppm of Mn, and 1.75 ppm of Cr. Group 3 received a 25% extra supplement to achieve a diet containing 87.50 ppm of Zn, 21.87 ppm of Cu, 81.25 ppm of Mn, and 2.18 ppm of Cr. The experiment was carried out for a total of 180 days. According to the findings, there was no significant impact of specific trace minerals supplementation on the animals' body weight, morphometric parameters, dry matter intake, average daily gain, nutritional value, digestibility and nitrogen retention. However, higher levels of Zn, Cu, and Mn led to increased (p < 0.05) total retention, while Cr retention remained the same. Serum mineral concentrations of Zn, Cu, and Mn increased significantly (p < 0.05) in G2 and G3 compared to the G1 group while Ca, P, and Cr had no significant change. Blood plasma glucose, albumin, globulin, and total protein showed no significant differences. Plasma alkaline phosphatase activity improved significantly (p < 0.05) in G2 and G3 but alanine transaminase, aspartate aminotransferase, hemoglobin, hematocrit, and IGF-1 remained unchanged. Superoxide dismutase activity, ferric-reducing antioxidant power, and total immunoglobulin concentration increased significantly (p < 0.05) in G2 and G3 groups, however, catalase activity and IgG count did not change among the groups. Mineral-supplemented groups (G2 and G3) showed a significant change (p < 0.05) in testosterone production during the 120th and the 180th day of the trial. Scrotal circumference and temperature gradient of the scrotal surface did not show any significant change. Supplementing growing bull calves with specific trace minerals above the basal level (70, 17.50, 65 and 1.75 ppm of Zn, Cu, Mn and Cr) has no direct beneficial effect on the growth parameters but can have positive effects on their antioxidant status, immunity and reproductive development as the related blood parameters were positively affected.

Development of a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for characterizing pomegranate extract pharmacokinetics in humans.

Pomegranate extracts standardized to punicalagins are a rich source of ellagitannins including ellagic acid (EA). Recent evidence suggests that gut microbiota-derived urolithin (Uro) metabolites of ellagitannins are pharmacologically active. Studies have evaluated the pharmacokinetics of EA, however, little is known about the disposition of urolithin metabolites (urolithin A (UA) and B (UB)). To address this gap, we developed and applied a novel ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) assay for the characterization of EA and Uro oral pharmacokinetics in humans. Subjects (10/cohort) received a single oral dose (250 or 1000 mg) of pomegranate extract (Pomella® extract) standardized to contain not less than 30 % punicalagins, < 5 % EA, and not less than 50 % polyphenols. Plasma samples, collected over 48 h, were treated with ß-glucuronidase and sulfatase to permit comparison between unconjugated and conjugated forms of EA, UA and UB. EA and urolithins were separated by gradient elution (acetonitrile/water, 0.1 % formic acid) using a C18 column connected to a triple quadrupole mass spectrometer operating in the negative mode. Conjugated EA exposure was ∼5-8-fold higher than unconjugated EA for both dose groups. Conjugated UA was readily detectable beginning ∼8 h post-dosing, however, unconjugated UA was detectable in only a few subjects. Neither form of UB was detected. Together these data indicate EA is rapidly absorbed and conjugated following oral administration of Pomella® extract. Moreover, UA's delayed appearance in the blood, primarily in the conjugated form, is consistent with gut microbiota-mediated metabolism of EA to UA, which is then rapidly converted to its conjugated form.

Effect of synbiotics on growth performance, gut health, and immunity status in pre-ruminant buffalo calves.

Synbiotics are employed as feed additives in animal production as an alternate to antibiotics for sustaining the gut microbiota and providing protection against infections. Dairy calves require a healthy diet and management to ensure a better future for the herd of dairy animals. Therefore, the present study was carried out to investigate the effect of synbiotics formulation on growth performance, nutrient digestibility, fecal bacterial count, metabolites, immunoglobulins, blood parameters, antioxidant enzymes and immune response of pre-ruminant Murrah buffalo calves. Twenty-four apparently healthy calves (5 days old) were allotted into four groups of six calves each. Group I (control) calves were fed a basal diet of milk, calf starter and berseem with no supplements. Group II (SYN1) calves were fed with 3 g fructooligosaccharide (FOS) + Lactobacillus plantarum CRD-7 (150 ml). Group III (SYN2) calves were fed with 6 g FOS + L. plantarum CRD-7 (100 ml), whereas calves in group IV (SYN3) received 9 g FOS + L. plantarum CRD-7 (50 ml). The results showed that SYN2 had the highest (P < 0.05) crude protein digestibility and average daily gain compared to the control. Fecal counts of Lactobacilli and Bifidobacterium were also increased (P < 0.05) in supplemented groups as compared to control. Fecal ammonia, diarrhea incidence and fecal scores were reduced in treated groups while lactate, volatile fatty acids and antioxidant enzymes were improved compared to the control. Synbiotic supplementation also improved both cell-mediated and humoral immune responses in buffalo calves. These findings indicated that synbiotics formulation of 6 g FOS + L. plantarum CRD-7 in dairy calves improved digestibility, antioxidant enzymes, and immune status, as well as modulated the fecal microbiota and decreased diarrhea incidence. Therefore, synbiotics formulation can be recommended for commercial use in order to achieve sustainable animal production.

Simultaneous Monitoring and Decontamination of Pesticide Residues in Phytomedicine-Enriched Betel Leaf Utilizing QuEChERS-GC-MS/MS Technology to Safeguard Public Health.

BACKGROUND: The presence of undesirable substances, including pesticides (xenobiotics) in betel leaf (Piper betel), is a great concern for consumers because it is chewed and consumed directly. To protect the consumer's health, a modified QuEChERS method for monitoring purposes and subsequent decontamination process has been developed. OBJECTIVE: The goal of this work was to establish a multi-residue analytical method for monitoring nonpermitted organophosphorus pesticide residues in betel leaf, as well as cost-effective cleaning strategies. METHOD: The homogenized 15 g samples (20 betel leaf samples collected in West Bengal, India) were extracted with a modified QuEChERS method using acetonitrile, reconstituted to acetone, and finally analyzed by GC-MS/MS. Possible decontamination techniques (such as tap water washing, 2% saltwater washing, and lukewarm water washing) were evaluated. RESULTS: The limit of detection ranged from 0.003 to 0.005 mg/kg, and limit of quantification was 0.01 mg/kg. Recoveries ranged from 80 to 120% with RSDr 9%. One sample was found to contain three pesticides 4 to 7 times higher than MRLs. Suggested decontamination methods allowed reducing toxic traces below European limits. CONCLUSIONS: The suggested approach is useful for determining pesticide residues in betel leaves quickly. Traditional techniques of processing betel leaves may reduce pesticide residues below regulatory limits. HIGHLIGHTS: A multi-residue method and decontamination of pesticides in betel leaf using QuEChERS-GC-MS/MS technology with satisfactory method performance was achieved. Domestic decontamination techniques have a high efficacy in reducing pesticide residues from betel leaves, making them safe for human consumption.

In Vitro Metabolism and CYP-Modulating Activity of Lavender Oil and Its Major Constituents.

The application of essential oils has historically been limited to topical (massage therapy) and inhalational (aromatherapy) routes of administration. More recently, however, evaluation of the therapeutic effects of essential oils has expanded to include the oral route of administration, which increases the herb-drug interaction potential. The purpose of this study was to evaluate the herb-drug interaction potential of lavender essential oil and two of its primary phytoactive constituents, namely linalool and linalyl acetate. The metabolic stability of linalool and linalyl acetate was determined in human liver microsomes (HLM) and S9 fractions by quantitative analysis using UPLC-MS/MS system. Linalool was metabolically unstable in HLM and S9 fractions with an intrinsic clearance of 31.28 mL·min-1·kg-1, and 7.64 mL·min-1·kg-1, respectively. Interestingly, it was observed that linalyl acetate converted to linalool both in HLM and S9 fractions. Lavender oil showed weak inhibitory effect on the catalytic activity of CYP3A4 and CYP1A2 enzymes (IC50 12.0 and 21.5 µg/mL). Linalyl acetate inhibited CYP3A4 (IC50 4.75 µg/mL) while linalool did not show any inhibitory effect on any of the enzymes. The lavender oil and its constituents did not activate PXR to a considerable extent, and no activation of AhR was observed, suggesting a lack of potential to modify the pharmacokinetic and pharmacodynamic properties of conventional medications if used concurrently.

Development of a GC/Q-ToF-MS Method Coupled with Headspace Solid-Phase Microextraction to Evaluate the In Vitro Metabolism of ß-Caryophyllene.

Sample preparation remains both a challenging and time-consuming process in the field of bioanalytical chemistry. Many traditional techniques often require multi-step processes, which can introduce additional errors to the analytical method. Given the complexity of many biological matrices, thorough analyte extraction presents a major challenge to researchers. In the present study, a headspace solid-phase microextraction (HS-SPME) coupled with a GC/Q-ToF-MS method, was developed to quantify in vitro metabolism of ß-caryophyllene by both human liver microsome (HLM) and S9 liver fractions. Validation of the method was demonstrated both in terms of linearity (R2 = 0.9948) and sensitivity with a limit of detection of 3 ng/mL and a limit of quantitation of 10 ng/mL. In addition, the method also demonstrated both inter- and intra-day precision with the relative standard deviation (RSD) being less than 10% with four concentrations ranging from 50-500 ng/mL. Since this method requires no solvents and minimal sample preparation, it provides a rapid and economical alternative to traditional extraction techniques. The method also eliminates the need to remove salts or buffers, which are commonly present in biological matrices. Although this method was developed to quantify in vitro metabolism of one analyte, it could easily be adapted to detect or quantify numerous volatiles and/or semi-volatiles found in biological matrices.

Impact of Moringa oleifera on rumen fermentation and methane emission under in vitro condition.

Exploring innovative methods to provide essential nutrients and reducing ruminant greenhouse gas emission is crucial for animal production and diminishing global warming. This study was conducted to examine the efficacy of Moringa oleifera leaves (ML) in ruminants at 0%, 5%, 10%, 15%, 20%, 30% and 40% level in different roughage (R) and concentrate (C) (80R:20C, 70R:30C and 60R:40C) under in vitro conditions. Chemical composition of ML, concentrate mixture and berseem were estimated. Rumen fermentation parameters of male goat kids viz., total gas production, CH4, true dry matter digestibility (TDMD), organic matter digestibility (TOMD), partial fraction (PF), microbial biomass (MBP), ammonia (N), acetate, propionate, butyrate and acetate propionate ratio were observed under in vitro conditions. Results revealed that crude protein, organic matter and ethyl ether content were higher in ML as compared to concentrate mixture and berseem. Magnesium and iron content were also higher in ML as compared to concentrate and berseem. Total gas production, digestibility of DM and OM, MBP, acetate and propionate level were improved (P < 0.05) upto 10-20% replacement. In contrast, decreased in CH4 (%) and CH4 (mL/100 mg dDM) was noted with increased levels of ML incorporation. There was no change observed in ammonia, acetate: propionate ratios at all the three planes of nutrition. In this study, it is concluded that mixing Moringa oleifera leaves in feed can be used as protein supplement and reduce the methane emission without causing any effect on digestibility and rumen fermentation parameters. However, ML can be suggested for widespread practice to attain the sustainable animal production (10-20%) and to alleviate the global warming.

Iodine Supplementation Improved Antioxidant Status, Hormonal Status, Sexual Behavior, and Semen Production Performance of Bos indicus Bulls Under Tropical Climatic Condition.

Iodine is anessential micronutrient that plays a crucial role in male reproduction (sexual behavior and semen production performance) by modulating thyroid function and the antioxidant status of the animal. Nonetheless, in Bos indicus bulls, a thorough evaluation of the effects of dietary iodine supplementation on antioxidant status, seminal quality parameters, and its interaction with other minerals is not documented. Twelve Bos indicus (Sahiwal) bulls were distributed into three groups (n = 4 in each group) viz. T1 (control), T2, and T3 and fed diets containing 0.250, 0.375, and 0.500 ppm iodine/ kg dry matter intake, corresponding to 0%, 50%, and 100% higher than ICAR (2013) recommendations, respectively. The experimental feeding was carried out for 60 days and the effects on nutrient utilization, hormonal and antioxidant status, and sperm function tests were investigated. Results revealed that body weight, dry matter intake, and nutrient digestibility remained unaffected by dietary supplementation of iodine. Testosterone and thyroxine hormone concentrations were improved (p<0.05) in T2 and T3 groups. Blood and seminal iodine content were also higher (p<0.05) in both the supplemented groups (T2 and T3). Sperm functions viz. viability, physical membrane integrity, acrosomal integrity, motility, and mitochondrial membrane potential were improved (p<0.05) due to iodine supplementation. Furthermore, lipid peroxidation and membrane scrambling in spermatozoa were reduced (p<0.05) in T2 and T3 groups. Blood antioxidant status (total antioxidant activity and GPx levels) was improved (p<0.05) in T2 and T3. Sexual behavior was also improved (p<0.05) in iodine-supplemented groups. Hence, it can be concluded that iodine supplementation at the dose rate of 0.500 ppm in the Bos indicus bull diet is beneficial in improving hormonal status, antioxidant status, and semen quality.

Impact of Moringa oleifera Leaves on Nutrient Utilization, Enteric Methane Emissions, and Performance of Goat Kids.

The development of different innovative feed resources for livestock is important to provide the essential nutrients and diminish the emission of greenhouse gases. The purpose of the present experiment was to study the response of replacing concentrate with Moringa oleifera leaves in terms of the nutrient intake, digestibility, enteric methane emissions, and performance of goat kids with a berseem-fodder-based diet under different roughage (R)-to-concentrate (C) ratios. Twenty-four goat kids (3 months of age) were distributed into four groups of six animals each, using a randomized block design (RBD). Kids of Group I (control) were fed a basal diet with 70R:30C without any tree leaf supplementation. Group II kids were fed with 60R:40C, where 10% of the concentrate mix was replaced with Moringa leaf (ML powder). In Group III, kids were fed with 70R:30C with 20% ML replacement. In Group IV, kids were fed with 80R:20C with 20% ML replacement. A metabolic trial was conducted after 180 days of feeding to assess the impact of ML on blood metabolites, antioxidant status, immunity parameters, and enteric methane emissions. The results revealed that dry matter digestibility, organic matter, and NDF were better (p < 0.05) in ML-treated kids (GII and GIII) compared to GI. Feed conversion and average daily gain were also enhanced (p < 0.05) in the treated groups as compared to controls. Total blood protein and albumin were increased in GII and GIII kids compared to GI. Plasma cholesterol levels were decreased (p < 0.001) in GII, GIII, and GIV as compared to GI. Glutathione peroxidase, catalase, and superoxide dismutase enzyme activities were also enhanced in GII, GIII, and GIV compared to controls. ML supplementation improved cell-mediated immunity and humoral immunity responses in goat kids. Enteric methane emissions decreased in the treated groups as compared to the controls. Moringa oleifera leaf may be used up to the level of 10−20% in concentrate mixes to improve digestibility, blood biochemical parameters, immunity status, and antioxidant activity in goat kids. Supplementation of ML not only enhanced the digestion and health of goat kids, but also decreased their methane emissions.

Development of a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for characterizing caffeine, methylliberine, and theacrine pharmacokinetics in humans.

Coffea liberica possesses stimulant properties without accumulating the methylxanthine caffeine. The basis for this peculiar observation is that methylurates (e.g., theacrine and methylliberine) have replaced caffeine. The stimulant properties of methylurates, alone and in combination with caffeine, have recently been investigated. However, human pharmacokinetics and LC-MS/MS methods for simultaneous measurement of methylxanthines and methylurates are lacking. To address this deficiency, we conducted a pharmacokinetic study in which subjects (n = 12) were orally administered caffeine (150 mg), methylliberine (Dynamine™, 100 mg), and theacrine (TeaCrine®, 50 mg) followed by blood sampling over 24 h. Liquid-liquid extraction of plasma samples containing purine alkaloids and internal standard (13C-Caffeine) were analyzed using a C18 reversed-phase column and gradient elution (acetonitrile and water, both containing 0.1% formic acid). A Waters Xevo TQ-S tandem mass spectrometer (positive mode) was used to detect caffeine, methylliberine, theacrine, and IS transitions of m/z 195.11 â†’ 138.01, 225.12 â†’ 168.02, 225.12 â†’ 167.95, and 198.1 â†’ 140.07, respectively. The method was validated for precision, accuracy, selectivity, and linearity and was successfully applied to characterize the oral pharmacokinetics of caffeine, methylliberine, and theacrine in human plasma. Successful development and application of LC-MS/MS-based methods such as ours for the simultaneous measurement of methylxanthines and methylurates are essential for the characterization of potential pharmacokinetic and pharmacodynamic interactions.

Effects of monensin feeding on performance, nutrient utilisation and enteric methane production in growing buffalo heifers.

Murrah buffalo heifers (live weight 135 ± 17 kg) were fed a total mixed ration without supplementation (CON), or supplemented with sodium monensin (MON; Rumensin® 200, Elanco Animal Health, Brazil) @ 0.6 mg/kg of body weight for 90 days. Nutrient digestibility and nitrogen retention were estimated during the mid-experiment, and enteric methane production was measured by sulphur hexafluoride tracer technique for consecutive-5 days after the digestion trial. The dry matter (DM) and nutrient intake were not affected but DM intake expressed as percent of body weight was decreased by monensin supplementation (3 vs 2.7% for CON and MON, respectively). The crude protein digestibility was higher for MON whereas, digestibility of other nutrients was not affected. Nitrogen retention (+ 4.59 g/day) and daily body weight gain (+ 56 g/day) were greater for MON-fed heifers without any significant effect on nitrogen intake and nitrogen excretion through faeces and urine. Daily enteric methane production was reduced by 12.61% but the treatments did not differ significantly. Methane emission expressed as gram per unit of DM, organic matter and digestible DM intake was lower for MON than CON and methane conversion rate (Ym) % of GE and ME intake was also decreased by 8-9%. On day 60, blood glucose level was increased and urea nitrogen was decreased in MON-fed heifers. This study indicated that monensin supplementation at 0.6 mg/kg body weight in growing heifers improved daily gain and feed efficiency while it reduced enteric methane production which can reduce feedlot time and consequent life time CH4 production.

Growth performance, and enteric and manure greenhouse gas emissions from Murrah calves fed diets with different forage to concentrate ratios.

The present study investigated the effects of different dietary forage to concentrate ratios on animal performance, and enteric and manure greenhouse gas emissions in growing calves. Fifteen male Murrah calves (153.5 ± 18.17 kg; 6 to 12 months) were randomly assigned to 3 dietary treatments and fed corn fodder, wheat straw and concentrate in 3 different proportions: 20:60:20 (C20); 20:40:40 (C40) and 10:30:60 (C60), for a period of 120 days. Increasing dietary concentrate proportion had no significant (P > 0.05) effect on dry matter intake (DMI) but increased crude protein (CP) and total digestible nutrient intake (P < 0.05). Average daily gain and feed conversion efficiency were significantly higher (P < 0.05) for C60 compared with C20 and for C40, these did not differ with C20 and C60 (P > 0.05). The apparent digestibility of dry matter, organic matter and CP were higher (P < 0.05), but acid detergent fiber digestibility was lower (P < 0.05) for C60 compared with C20 whereas, ether extract and neutral detergent fiber digestibilities were not affected (P > 0.05). Daily methane (CH4) emission (g/d), CH4 energy loss (MJ/d) and CH4 yield (CH4 g/kg organic matter intake [OMI], CH4 g/kg digestible OMI, and CH4 % of metabolizable energy intake) were significantly higher for C20 compared with C60 (P < 0.05). Methane yield as g/kg DMI although lower for C60 compared with C20 but the difference was not significant (P > 0.05). Manure CH4 (g/kg DMI) and nitrous oxide (N2O mg/kg nitrogen) emissions were not affected (P > 0.05), but N2O emission on mg/kg DM basis was significantly higher (P < 0.05) from the manure of calves fed C60 than that for C20. Thus, increasing dietary concentrate proportion improved animal performance, and reduced enteric CH4 emission (g/day) without any significant effect on manure N2O (mg/kg nitrogen) and CH4 emissions.

Coadministration of Polymeric Conjugates of Docetaxel and Cyclopamine Synergistically Inhibits Orthotopic Pancreatic Cancer Growth and Metastasis.

PURPOSE: The aim of this study was to determine whether co-administration of hedgehog (Hh) pathway inhibitor cyclopamine (CYP) and microtubule stabilizer docetaxel (DTX) as polymer-drug conjugates, methoxy poly(ethylene glycol)-block-poly(2-methyl-2-carboxyl-propylenecarbonate-graft-dodecanol-graft-cyclopamine) (P-CYP) and methoxy poly(ethylene glycol)-block-poly(2-methyl-2-carboxyl-propylene carbonate-graft-dodecanol-graft-docetaxel) (P-DTX) could synergistically inhibit orthotopic pancreatic tumor growth in NSG mice. METHODS: P-DTX and P-CYP were synthesized from mPEG-b-PCC through carbodiimide coupling reaction and characterized by 1H-NMR. The micelles were prepared by film hydration and particle size was measured by dynamic light scattering (DLS). Cytotoxicity, apoptosis and cell cycle analysis of P-DTX and P-CYP were evaluated in MIA PaCa-2 cells. In vivo efficacy of P-DTX and P-CYP were evaluated in NSG mice bearing MIA PaCa-2 cells derived orthotopic pancreatic tumor. RESULTS: P-CYP and P-DTX self-assembled into micelles of <90 nm and their combination therapy efficiently inhibited the proliferation of MIA PaCa-2 cells, induced apoptosis and cell cycle arrest at M-phase more efficiently than P-CYP and P-DTX monotherapies. Furthermore, the combination therapy of P-CYP and P-DTX significantly reduced Hh component expression compared to P-CYP alone as determined by Western blot analysis. Lastly, the combination therapy induced greater inhibition of orthotopic pancreatic tumor growth in NSG mice compared to their monotherapies. CONCLUSION: Combination of polymer conjugated anticancer drug (P-DTX) with polymer conjugated Hh inhibitor (P-CYP) enhanced pancreatic cancer cell killing, apoptosis as well as in vivo tumor growth inhibition with no obvious toxicities.

EGFR-Targeted Cationic Polymeric Mixed Micelles for Codelivery of Gemcitabine and miR-205 for Treating Advanced Pancreatic Cancer.

Gemcitabine (GEM), a first-line chemotherapy for pancreatic cancer undergoes rapid metabolism and develops chemoresistance after repeated administration. We previously demonstrated that the combination of GEM and miR-205 provides an effective therapeutic strategy to sensitize GEM-resistant pancreatic cancer cells. Since epidermal growth factor receptor (EGFR) is overexpressed in pancreatic cancer cells, in this study, we aimed to deliver mixed micelles containing GEM and miR-205 decorated with EGFR-targeting cetuximab (C225) monoclonal antibody for targeted therapy. Cetuximab C225 was conjugated to malemido-poly(ethylene glycol)-block-poly(2-methyl-2-carboxyl-propylene carbonate-graft-dodecanol (C225-PEG-PCD) to prepare mixed micelles with mPEG-b-PCC-g-GEM-g-DC-g-TEPA for targeted codelivery of GEM and miR-205. This mixed micelle formulation showed a significant enhancement in EGFR-mediated cellular uptake in GEM-resistant MIA PaCa-2R cells. Further, an enhanced tumor accumulation of C225-micelles conjugated with near-infrared fluorescent Cy7.5 dye and Dy677-labeled miR-205 in orthotopic pancreatic tumor bearing NSG mice was evident after systemic administration. In addition, inhibition of tumor growth was also observed with increased apoptosis and reduced EMT after treatment with C225-micelles containing GEM and miR-205. Therefore, we believe that the targeted delivery of GEM and miR-205 in combination could be a novel strategy for treating advanced pancreatic cancer.

Chemosensitization and inhibition of pancreatic cancer stem cell proliferation by overexpression of microRNA-205.

Treatment of pancreatic cancer with gemcitabine (GEM) is limited due to its rapid plasma metabolism and development of chemoresistance. MicroRNA (miRNA) regulates cancer stem cell (CSC) maintenance and induces chemoresistance in cancer cells. In this study, we observed differential downregulation of miR-205 (miR-205-5p) in human pancreatic cancer tissues and cells. Compared to GEM-sensitive MIA PaCa-2 cells, miR-205 was highly downregulated in GEM-resistant MIA PaCa-2R cells. Lentivirus-mediated overexpression of miR-205 inhibits MIA PaCa-2R cell proliferation after GEM-treatment. Further investigation confirmed that miR-205 alone significantly reduces the proliferation of CSCs and tumor growth in mouse models. However, miR-205 in combination with GEM was more efficient in reducing the proliferation of CSCs and 3D spheroids. Moreover, miR-205 overexpressing MIA PaCa-2R cells induced orthotopic tumor growth was significantly inhibited after intravenous administration of GEM-conjugated methoxy poly(ethylene glycol)-block-poly(2-methyl-2-carboxyl-propylene carbonate)-graft-gemcitabine-graft-dodecanol (mPEG-b-PCC-g-GEM-g-DC) (mPEG-b-PCC-g-GEM-g-DC) polymeric micelles. Also, a reduction in CSCs, EMT and chemoresistance markers was observed in miR-205 overexpressing MIA PaCa-2R cells. Immunohistochemical analysis of orthotopic tumors showed a decrease in drug resistance protein caveolin-1 and cell proliferation marker Ki-67 in combination treatment. Overall, our findings suggest that miR-205 resensitizes GEM-resistant pancreatic cancer cells to GEM and acts as a tumor suppressor miRNA.

Design, Synthesis and Biological Evaluation of novel Hedgehog Inhibitors for treating Pancreatic Cancer.

Hedgehog (Hh) pathway is involved in epithelial-mesenchymal transition (EMT) and cancer stem cell (CSC) maintenance resulting in tumor progression. GDC-0449, an inhibitor of Hh pathway component smoothened (Smo) has shown promise in the treatment of various cancers including pancreatic cancer. However, the emergence of resistance during GDC-0449 treatment with numerous side effects limits its use. Therefore, here we report the design, synthesis and evaluation of novel GDC-0449 analogs using N-[3-(2-pyridinyl) phenyl] benzamide scaffold. Cell-based screening followed by molecular simulation revealed 2-chloro-N 1-[4-chloro-3-(2-pyridinyl)phenyl]-N 4,N 4-bis(2-pyridinylmethyl)-1,4-benzenedicarboxamide (MDB5) as most potent analog, binding with an extra interactions in seven-transmembrane (7-TM) domain of Smo due to an additional 2-pyridylmethyl group than GDC-0449. Moreover, MDB5 was more efficient in inhibiting Hh pathway components as measured by Gli-1 and Shh at transcriptional and translational levels. Additionally, a significant reduction of ALDH1, CD44 and Oct-3/4, key markers of pancreatic CSC was observed when MIA PaCa-2 cells were treated with MDB5 compared to GDC-0449. In a pancreatic tumor mouse model, MDB5 containing nanoparticles treated group showed significant inhibition of tumor growth without loss in body weight. These evidence highlight the enhanced Hh pathway inhibition and anticancer properties of MDB5 leaving a platform for mono and/or combination therapy.