Effects of a Phytogenic Supplement Containing Olive By-Product and Green Tea Extracts on Growth Performance, Lipid Metabolism, and Hepatic Antioxidant Capacity in Largemouth Bass (Micropterus salmoides) Fed a High Soybean Meal Diet.

A 10-week growth trial was conducted to investigate the effects of a phytogenic feed additive (PFA) containing olive by-products and green tea extracts supplemented to a reduced fishmeal/high soybean meal diet on the growth performance, hepatic antioxidant capacity, lipid metabolism, and liver health of largemouth bass (Micropterus salmoides). Three experimental diets were tested: (1) a control high fishmeal (40%) and low soybean meal (15.57%) diet (named HFM), (2) a reduced fishmeal (30%) and high soybean meal (30.97%) diet (named HSB), and (3) a HSB diet supplemented with the PFA at 500 mg/kg (named HSB+P). Each diet was assigned to four replicate tanks, each containing 30 largemouth bass (initial body weight, IBW = 48.33 ± 0.01 g). The results showed that increasing the soybean meal content in the diet did not negatively affect growth performance, whereas supplementation with PFA significantly increased weight gain and specific growth rate of largemouth bass compared to both HFM and HSB groups. Reducing fishmeal and increasing soybean meal in the diet caused oxidative stress with a higher content of ROS in the liver. However, the hepatic antioxidant capacity was enhanced, with reduced ROS and increased GSH-Px levels in the HSB+P group. Moreover, the decrease of plasma TG, LDL-C, and LDL-C/TC, and downregulation of lipogenesis and cholesterol synthesis gene expression in liver, indicated that supplementation with the PFA improved fish lipid metabolism. Protein retention efficiency was also significantly increased in largemouth bass fed the diet with PFA supplementation, which regulated (enhanced) AKT-mTOR phosphorylation. These results clearly indicated that a PFA containing olive by-product and green tea extracts can positively improve growth performance, protein retention efficiency, antioxidant capacity, and lipid metabolism of largemouth bass fed a reduced fishmeal/high soybean meal diet.

Effects of High Starch and Supplementation of an Olive Extract on the Growth Performance, Hepatic Antioxidant Capacity and Lipid Metabolism of Largemouth Bass (Micropterus salmoides).

An 8-week feeding trial was conducted to investigate the effects of high-starch diets and the supplementation of an olive extract (OE) on the growth performance, liver health and lipid metabolism of largemouth bass (Micropterus salmoides). Four isonitrogenous and isolipidic diets were prepared: two basal diets containing low (9.0%) and high (14.4%) levels of starch (named as LS and HS), and 0.125% OE was supplemented to each basal diet (named LSOE and HSOE). The results show that high-starch diets had significant negative effects on growth performance, with lower FR, SGR and higher FCR, whereas OE significantly lowered FCR, determined by two-way ANOVA analysis. High-starch diets induced oxidative stress, inflammatory response and liver function injury, with significant increases in the content of plasmatic AKP, AST, ALT, hepatic SOD and MDA, and up-regulation of hepatic TNFα, IL1ß, and TGFß1 gene expression. In addition, a high-starch diet decreased the phosphorylation of AMPK and upregulated the expression of SREBP, together with higher hepatic liver lipid and HSI. The oxidative stress and lipid metabolism disorders indicate metabolic liver disease (MLD) of largemouth bass fed high-starch diets. Feeding on OE-supplemented diets increased the hepatic antioxidant capacity by decreasing the content of MDA and SOD. Fish fed the HSOE diet had an activated phosphorylation of JNK and decreased expression of pro-inflammatory IL1ß compared with those fed the HS diet, which strongly indicated that the degree of inflammatory responses was reduced after OE supplementation. Interestingly, this study demonstrated that OE regulates hepatic lipid metabolism in fish by inhibiting the expression of hepatic lipogenesis genes (ACC1 and FASN) and promoting lipolysis (ATGL) and ß-oxidation (CPT1α) to prevent TG accumulation. In conclusion, high-starch feed induced oxidative stress and lipid metabolic disorder of largemouth bass, while supplementation with OE improved its antioxidant capacity, anti-inflammatory responses and lipid metabolism. However, hepatic histopathological results suggested that OE supplementation could not completely repair the MLD caused by the high level of starch in largemouth bass.

Olive oil cake extract stabilizes the physiological condition of lipopolysaccharide-challenged piglets by reducing oxidative stress and inflammatory responses and modulating the ileal microbiome.

Olives are a rich source of compounds with antioxidant and anti-inflammatory activities. This study was designed to investigate whether a standardized olive cake extract was able to alleviate oxidative stress, inflammation and intestinal villus damage in a model of lipopolysaccharide (LPS)-challenged piglets. Thirty weaned piglets (6.9 ± 0.9 kg) were assigned to five groups using a randomized complete block design. Piglets were fed a basal diet before intraperitoneal (i.p.) injection of physiological saline (C); fed a basal diet alone (CL) or fed a basal diet plus an olive cake extract (OL), antibiotics (AL), or olive cake extract plus antibiotics (OAL) before i.p. injection of LPS. The feeding period lasted for 2 weeks. Piglets were euthanized 4 h after the LPS injection. Systemic oxidative and inflammatory status and intestinal morphology were evaluated. LPS challenge significantly lowered the serum levels of GSH-Px, SOD and ALB and increased the serum concentration of MDA, NO, LDH, ALT, AST, TNF-α, IL-6, DAO and D-xylose (P < 0.05), as extracted from the comparison of piglets in the C and CL groups. Intestinal morphology was altered in the duodenum and ileum, displaying that the CL group had significantly lower villus height (VH), higher crypt depth (CD) and lower VH/CD compared with the control group (P < 0.05). Moreover, feed supplementation was able to partially mitigate the negative effects of LPS challenge in all groups (OL, AL, and OAL), as evidenced by the significantly increased serum levels of GSH-Px, SOD, ALB and IL-10 and decreased concentration of MDA, NO, LDH, ALT, AST, TNF-α, IL-6, DAO and D-xylose, compared with the CL group (P < 0.05). Alterations in intestinal morphology were also prevented and the OL, AL, and OAL groups had significantly lower CD and higher VH/CD compared with the CL group (P < 0.05), both in the ileum and duodenum. Furthermore, the positive effect in the relative abundance of intestinal Lactobacillus and Clostridium at the genus level was also observed for the OL group compared to the CL group. In summary, dietary supplementation with an olive cake extract stabilized the physiological condition of piglets subjected to an acute LPS challenge by reducing oxidative stress and the inflammatory status, improving intestinal morphology and increasing the abundance of beneficial intestinal bacteria. This trial was registered at Zhejiang University (http://www.lac.zju.edu.cn) as No. ZJU20170529.

Encapsulated Mixture of Methyl Salicylate and Tributyrin Modulates Intestinal Microbiota and Improves Growth Performance of Weaned Piglets.

Tributyrin and essential oils have been used as alternatives to antimicrobials to improve gut health and growth performance in piglets. This study was to evaluate the effects of a dietary supplement with two encapsulated products containing different combinations of tributyrin with oregano or with methyl salicylate on growth performance, serum biochemical parameters related to the physiological status, intestinal microbiota and metabolites of piglets. A total of 108 weaned crossbred piglets (Yorkshire × Landrace, 21 ± 1 d, 8.21 ± 0.04 kg) were randomly divided into three groups. Piglets were fed with one of the following diets for 5 weeks: a basal diet as the control (CON); the control diet supplemented with an encapsulated mixture containing 30% of methyl salicylate and tributyrin at a dosage of 3 kg/t (CMT); and the control diet supplemented with an encapsulated mixture containing 30% of oregano oil and tributyrin at a dosage of 3 kg/t (COT). At the end of the feeding trial, six piglets from each group were slaughtered to collect blood and gut samples for physiological status and gut microbiological analysis. The study found that the CMT group was larger in feed intake (FI) (p < 0.05), average daily gain (ADG) (p = 0.09), total protein (TP), albumin (ALB), glutathione peroxidase (GSH-PX) (p < 0.05), blood total antioxidant capacity (T-AOC) (p < 0.05), and crypt depth in the ileum (p < 0.05) compared with the CON group. The genus abundance of Tissierella and Campylobacter in the CMT group was significantly decreased compared with the CON group. The CMT group also resulted in significantly higher activity in amino acid metabolism and arginine biosynthesis compared with the CON group. The COT group was larger in T-AOC, and the genus abundance of Streptophyta and Chlamydia was significantly increased in the ileum compared with the CON group. Data analysis found a significantly high correlation between the genus abundance of Chlamydia and that of Campylobacter in the ileum. The genus abundance of Campylobacter was also positively correlated with the sorbitol level. In general, the results indicated that the supplementation of both encapsulated mixtures in diet of weaned piglets could improve the animal blood antioxidant capacity. Additionally, the encapsulated mixture of methyl salicylate plus tributyrin improved the growth performance and resulted in certain corresponding changes in nutrient metabolism and in the genus abundance of ileum microbial community.

Association of leptin genotype with growth performance, adipocyte cellularity, meat quality, and fatty acid profile in beef steers fed flaxseed or high-oleate sunflower seed diets with or without triticale dried distiller's grains.

Leptin genotypes can be identified as homozygous normal (CC), homozygous mutant (TT), and heterozygous (CT) based on a single-nucleotide polymorphism in exon 2 of the leptin gene, which has been associated with feed intake and fat deposition in cattle. The experiment was designed as 2 × 2 × 2 factorial with three main factors: (1) genotype (CT or TT) and diets fed 2) with or without triticale dried distiller's grains with solubles (DDG), and 3) with either flaxseed (FS) or high-oleate sunflower seed (SS). Evaluations included growth performance, subcutaneous fat deposition, adipocyte cellularity, meat quality, and fatty acid (FA) profile of various depots. Beef steers (n = 40, 459 ± 31 kg) of either CT or TT genotypes were housed in individual pens with ad libitum access to one of the four diets: 75% steam-rolled barley + 10% barley silage with 10% FS or SS (non-DDG diets, NDG) and 46.5% barley + 10% barley silage + 30% DDG, with 8.5% FS or SS, all on a dry matter basis. Growth performance, ultrasound subcutaneous fat thickness, rib eye area (REA), and plasma FA were measured prior to and during the finishing period. At slaughter, samples of subcutaneous fat, perirenal fat, and Longissimus thoracis (LT) muscle were collected for FA analysis and carcass and meat quality were measured. Compared with CT cattle, TT tended to have less (P = 0.06) C18:2-c9,t11 (rumenic acid) in plasma and subcutaneous fat and a greater proportion (P < 0.05) of C18:0 in subcutaneous, perirenal, and LT fat. Cattle with TT genotype also tended (P < 0.1) to have more total saturated and less unsaturated (USFA) and monounsaturated fats (MUFA) and had less (P = 0.04) linoleic acid in LT. Ultrasound fat thickness, REA, and average diameter of adipocytes in subcutaneous fat at 12 wk were not affected (P > 0.39) by genotype. Generally, carcass and meat quality were similar (P > 0.1) among diets, although adding FS tended to increase (P = 0.06) total USFA of subcutaneous fat including omega-3 FA (P < 0.001). For the high-fat diets evaluated, CT cattle would have more potential to produce beef with enhanced health benefits than would TT cattle.

Effects of Dietary Supplementation with Combination of Tributyrin and Essential Oil on Gut Health and Microbiota of Weaned Piglets.

The aim of this study was to determine the effects of dietary inclusion of the combination of tributyrin with oregano or methyl salicylate as a substitute to antibiotics on gut health and microbiota of piglets. A total of 48 weaned crossbred piglets (Duroc × Large White × Landrace, 8.79 ± 0.97 kg, 21 ± 1 d) were randomly allocated to four experimental groups and fed for 4 weeks: the basal diet (Con); the control plus antibiotics (AB); the control plus oregano and tributyrin (OT); and the control plus methyl salicylate and tributyrin (MT). Although a numerical improvement on feed intake, weight gain and feed conversion ratio was observed in the OT and MT as well as the AB group, the difference was not significant (p > 0.05). The OT and MT groups were larger in villus height in the duodenum compared to the Con (p < 0.05), and were larger in relative abundance of Firmicutes/Bacaeroidesin the intestine compared to Con and AB groups (p < 0.01). The amount of major different metabolites was 6, 8 and 8 for the AB, OT and MT groups when compared to the Con, respectively. In conclusion, as a substitute for antibiotics the inclusion of the combination of tributyrin with either oregano or methyl salicylate to the diet of weaned piglets improved the intestinal morphological structure and altered intestinal microbiota and metabolites, which were beneficial to the animal health.

Impact of a phytogenic feed additive on growth performance, feed intake, and carcass traits of finishing steers.

The purpose of this study was to evaluate the effect of a phytogenic feed additive (Digestarom [DA]; Biomin, Getzersdorf, Austria) on growth performance, feed intake, carcass traits, fatty acid composition, and liver abscesses of finishing steers. One hundred twenty Angus × Charolais crossbred steers (488 ± 26.5 kg) were used in a 110-d feeding experiment. Steers were blocked by weight and randomly assigned to 12 pens with 10 steers per pen. Each pen was allocated to one of three diets. Each diet contained 86.5% barley, 10.0% barley silage, and 3.5% vitamin and mineral supplement on a dry matter (DM) basis. The diets contained 0, 0.05, and 0.1 g DA/kg complete diet (DM basis), to achieve average daily DA intakes of 0 (control), 0.5 (LowDA), and 1.0 g (HighDA) per steer. Diets were prepared once daily and provided ad libitum. Two pens per treatment were equipped to record individual feed intake behavior. Steers were weighed every 28 d and carcass traits and liver scores were recorded at slaughter. Dry matter intake (average: 9.34 kg/d) did not differ (P > 0.05) among diets. Average daily gain tended to increase linearly as DA increased (control: 1.82; LowDA: 1.87; and HighDA: 1.95 kg/d; P < 0.09), but gain:feed ratio was not affected. Supplementation of DA affected longissimus muscle area quadratically (P = 0.05) with the largest area observed for LowDA. However, dressing percentage decreased linearly in response to increasing level of DA (P < 0.01). Total abscessed livers were not affected, whereas proportion of severe liver abscesses was numerically lower with DA (30.8% and 42.5% for LowDA and HighDA) compared to the control (50%).

Comparison on the fatty acid profiles of liver, subcutaneous fat and muscle from feedlot steers finished on diets supplemented with or without cinnamaldehyde or monensin.

BACKGROUND: Cinnamaldehyde (CIN) is the main active component of cinnamon (Cinnamomum cassia) oil and has been tested as alternative feed additive in cattle production. Little information was available on the effect of dietary CIN in comparison to monensin (MO) on beef fatty acid (FA) profile. This study analyzed FA profiles of liver, subcutaneous fat and pars costalis diaphragmatis (PCD) muscle obtained from steers (n = 70) finished on diets: control, a barley grain-silage feedlot diet; 330 mg/head.day MO; and 400, 800 or 1600 mg/head.day CIN treatments. RESULTS: Inclusion of MO or CIN did not affect total saturated, unsaturated, polyunsaturated FA and individual FA in the various tissues with exceptions that proportion of palmitic acid in PCD muscle was increased by 800 mg/steer.day CIN (P < 0.05). There were positive correlations (P < 0.05) on oleic, linoleic, conjugated linoleic acid (CLA)-c9,t11 and 18:1-t10 between the subcutaneous fat and PCD muscle, and on α-linolenic acid, CLA-c9,t11 and 18:1-t10 between PCD muscle and liver, whereas correlations on the FA between the subcutaneous fat and liver were not significant except for 18:1-t10 (P < 0.01). CONCLUSION: The results indicate that the supplementation of CIN and MO to feedlot diet has limited effect on beef FA profiles.

MicroRNAs in bovine adipogenesis: genomic context, expression and function.

BACKGROUND: MicroRNAs (miRNAs) are small non-coding RNAs found to regulate several biological processes including adipogenesis. Understanding adipose tissue regulation is critical for beef cattle as fat is an important determinant of beef quality and nutrient value. This study analyzed the association between genomic context characteristics of miRNAs with their expression and function in bovine adipose tissue. Twenty-four subcutaneous adipose tissue biopsies were obtained from eight British-continental crossbred steers at 3 different time points. Total RNA was extracted and miRNAs were profiled using a miRNA microarray with expression further validated by qRT-PCR. RESULTS: A total of 224 miRNAs were detected of which 155 were expressed in all steers (n = 8), and defined as the core miRNAs of bovine subcutaneous adipose tissue. Core adipose miRNAs varied in terms of genomic location (59.5% intergenic, 38.7% intronic, 1.2% exonic, and 0.6% mirtron), organization (55.5% non-clustered and 44.5% clustered), and conservation (49% highly conserved, 14% conserved and 37% poorly conserved). Clustered miRNAs and highly conserved miRNAs were more highly expressed (p < 0.05) and had more predicted targets than non-clustered or less conserved miRNAs (p < 0.001). A total of 34 miRNAs were coordinately expressed, being part of six identified relevant networks. Two intronic miRNAs (miR-33a and miR-1281) were confirmed to have coordinated expression with their host genes, transcriptional factor SREBF2 and EP300 (a transcriptional co-activator of transcriptional factor C/EBPα), respectively which are involved in lipid metabolism, suggesting these miRNAs may also play a role in regulation of bovine lipid metabolism/adipogenesis. Furthermore, a total of 17 bovine specific miRNAs were predicted to be involved in the regulation of energy balance in adipose tissue. CONCLUSIONS: These findings improve our understanding on the behavior of miRNAs in the regulation of bovine adipogenesis and fat metabolism as it reveals that miRNA expression patterns and functions are associated with miRNA genomic location, organization and conservation.

Elucidation of molecular mechanisms of physiological variations between bovine subcutaneous and visceral fat depots under different nutritional regimes.

Adipose tissue plays a critical role in energy homeostasis and metabolism. There is sparse understanding of the molecular regulation at the protein level of bovine adipose tissues, especially within different fat depots under different nutritional regimes. The objective of this study was to analyze the differences in protein expression between bovine subcutaneous and visceral fat depots in steers fed different diets and to identify the potential regulatory molecular mechanisms of protein expression. Subcutaneous and visceral fat tissues were collected from 16 British-continental steers (15.5 month old) fed a high-fat diet (7.1% fat, n=8) or a control diet (2.7% fat, n=8). Protein expression was profiled using label free quantification LC-MS/MS and expression of selected transcripts was evaluated using qRT-PCR. A total of 682 proteins were characterized and quantified with fat depot having more impact on protein expression, altering the level of 51.0% of the detected proteins, whereas diet affected only 5.3%. Functional analysis revealed that energy production and lipid metabolism were among the main functions associated with differentially expressed proteins between fat depots, with visceral fat being more metabolically active than subcutaneous fat as proteins associated with lipid and energy metabolism were upregulated. The expression of several proteins was significantly correlated to subcutaneous fat thickness and adipocyte size, indicating their potential as adiposity markers. A poor correlation (r=0.245) was observed between mRNA and protein levels for 9 genes, indicating that many proteins may be subjected to post-transcriptional regulation. A total of 8 miRNAs were predicted to regulate more than 20% of lipid metabolism proteins differentially expressed between fat depots, suggesting that miRNAs play a role in adipose tissue regulation. Our results show that proteomic changes support the distinct metabolic and physiological characteristics observed between subcutaneous and visceral adipose tissue depots in cattle.

Altered microRNA expression in bovine subcutaneous and visceral adipose tissues from cattle under different diet.

BACKGROUND: MicroRNAs (miRNAs) are a class of molecular regulators found to participate in numerous biological processes, including adipogenesis in mammals. This study aimed to evaluate the differences of miRNA expression between bovine subcutaneous (backfat) and visceral fat depots (perirenal fat) and the dietary effect on miRNA expression in these fat tissues. METHODOLOGY/PRINCIPAL FINDINGS: Fat tissues were collected from 16 Hereford×Aberdeen Angus cross bred steers (15.5 month old) fed a high-fat diet (5.85% fat, n = 8) or control diet (1.95% fat, n = 8). Total RNA from each animal was subjected to miRNA microarray analysis using a customized Agilent miRNA microarray containing 672 bovine miRNA probes. Expression of miRNAs was not equal between fat depots as well as diets: 207 miRNAs were detected in both fat depots, while 37 of these were found to be tissue specific; and 169 miRNAs were commonly expressed under two diets while 75 were diet specific. The number of miRNAs detected per animal fed the high fat diet was higher than those fed control diet (p = 0.037 in subcutaneous fat and p = 0.002 visceral fat). Further qRT-PCR analysis confirmed that the expression of some miRNAs was highly influenced by diet (miR-19a, -92a, -92b, -101, -103, -106, -142-5p, and 296) or fat depot (miR-196a and -2454). CONCLUSIONS/SIGNIFICANCE: Our results revealed that the miRNA may differ among adipose depots and level of fat in the diet, suggesting that miRNAs may play a role in the regulation of bovine adipogenesis.

Effects of wheat dried distillers' grains with solubles and cinnamaldehyde on in vitro fermentation and protein degradation using the Rusitec technique.

This study was conducted to evaluate the effect of wheat dried distillers' grains with solubles (DDGS) and cinnamaldehyde (CIN) on in vitro fermentation and microbial profiles using the rumen simulation technique. The control substrate (10% barley silage, 85% barley grain and 5% supplement, on dry matter basis) and the wheat DDGS substrate (30% wheat DDGS replaced an equal portion of barley grain) were combined with 0 and 300 mg CIN/l of culture fluid. The inclusion of DDGS increased (p < 0.05) the concentration of volatile fatty acids (VFA) and the molar proportion of acetate and propionate. Dry matter disappearance (p = 0.03) and production of bacterial protein (p < 0.01) were greater, whereas the disappearances of crude protein (CP) and neutral detergent fibre were less (p < 0.01) for the DDGS than for the control substrate. With addition of CIN, concentration of total VFA decreased and fermentation pattern changed to greater acetate and less propionate proportions (p < 0.01). The CIN reduced (p < 0.01) methane production and CP degradability. The copy numbers of Fibrobacter, Prevotella and Archaea were not affected by DDGS but were reduced (p < 0.05) by CIN. The results indicate that replacing barley grain by DDGS increased nutrient fermentability and potentially increase protein flows to the intestine. Supplementation of high-grain substrates with CIN reduced methane production and potentially increased the true protein reaching the small intestine; however, overall reduction of feed fermentation may lower the feeding value of a high-grain diet.

In situ identification of carboxymethyl cellulose-digesting bacteria in the rumen of cattle fed alfalfa or triticale.

A method was developed and used to arrest and stain reducing sugars (glucose) produced by bacteria with cell-surface-associated carboxymethyl cellulase (CMCase) and endoglucanase activities (CMC bacteria) in the rumen of cows fed alfalfa or triticale. Precipitation of silver oxide on the surface of individual cells was observed using cellulolytic bacterial pure cultures with known CMCase activity and rumen mixed cultures. The CMC bacteria in the liquid and solid fractions of the rumen digesta were identified using fluorescence in situ hybridization (FISH) with currently available and newly designed oligonucleotide probes. The CMC bacteria contributed between 8.2% and 10.1% to the total bacterial cell numbers. Most of the CMC bacteria (75.2-78.5%) could be identified by FISH probing. The known cellulolytic populations Ruminococcus flavefaciens, R. albus, and Fibrobacter succinogenes constituted 44.5-53.1% of the total. Other CMC bacteria identified hybridized with the probe Clo549 (11.2-23.0%) targeting members of an uncharacterized genus in Clostridia, the probe Inc852 (8.9-10.7%) targeting members of the family Incertae Sedis III and unclassified Clostridiales, and the probe But1243 (< 1%) designed against members of genus Butyrivibrio. Different forage feeds had no marked effects on the percentage abundances of these identified CMC bacteria. All appeared to be involved in cellulose degradation in the rumen of cows fed either alfalfa or triticale.

Inhibitory effect of antioxidant extracts from various potatoes on the proliferation of human colon and liver cancer cells.

Antioxidant extracts from 5 potato lines were evaluated for antioxidant activity, total phenolics, chlorogenic acid, anthocyanin content, and in vitro anticancer capacity. Analysis showed that Mexican wild species S. pinnatisectum had the highest antioxidant activity, total phenolic, and chlorogenic acid content. The proliferation of colon cancer and liver cancer cells was significantly inhibited by potato antioxidant extracts. The highest antiproliferative activity was observed in extracts of S. pinnatisectum and the lowest in Northstar. An inverse correlation was found between total phenolics and the EC(50) of colon cancer cell (R(2) = 0.9303), as well as liver cancer cell proliferation (R(2) = 0.8992). The relationship between antioxidant activity and EC(50) of colon cancer/liver cancer cell proliferation was significant (R(2) = 0.8144; R(2) = 0.956, respectively). A significant difference in inhibition of cancer cells (P < 0.01) existed between the 3 polyphenols: chlorogenic acid, pelargonidin chloride, and malvidin chloride, suggesting that chlorogenic acid was a critical factor in the antiproliferation of colon cancer and liver cancer cells.

Protection of mice against enterotoxigenic E. coli by immunization with a polyvalent enterotoxin comprising a combination of LTB, STa, and STb.

Currently available enterotoxigenic Escherichia coli (ETEC) vaccines are based on colonization factors and/or the heat-labile enterotoxin B subunit (LTB). However, the induction of antitoxic responses against heat-stable enterotoxin a (STa) and b (STb) has merit as these two poorly immunogenic toxins are frequently associated with ETEC strains. In this study, we genetically constructed a trivalent enterotoxin fusion protein (STa-LTB-STb, abbreviated to SLS) in an effort to develop a single toxoid containing these three enterotoxins for vaccination against ETEC. Mutagenesis at one disulfide-bridge-forming cysteine in STa led to a dramatic reduction in the STa toxicity of SLS; however, the fusion peptide retained the STb-associated toxicity. Immunization of mice with SLS protein elicited significant antibody responses to LTB, STa, and STb. Significantly, the mice antisera were able to neutralize the biological activity of both STa and STb. In the experiment to assess the protective effect of SLS immunization, the mortality of mice receiving SLS was significantly lower than their control cohorts (P < 0.01) after intraperitoneal challenge with ETEC. These results show that the trivalent fusion enterotoxin SLS has the potential to serve as a useful toxin-based vaccine against ETEC-induced diarrheal disease via a single immunogen.

Quantitative fluorescence in situ hybridization of microbial communities in the rumens of cattle fed different diets.

At present there is little quantitative information on the identity and composition of bacterial populations in the rumen microbial community. Quantitative fluorescence in situ hybridization using newly designed oligonucleotide probes was applied to identify the microbial populations in liquid and solid fractions of rumen digesta from cows fed barley silage or grass hay diets with or without flaxseed. Bacteroidetes, Firmicutes, and Proteobacteria were abundant in both fractions, constituting 31.8 to 87.3% of the total cell numbers. They belong mainly to the order Bacteroidales (0.1 to 19.2%), hybridizing with probe BAC1080; the families Lachnospiraceae (9.3 to 25.5%) and Ruminococcaceae (5.5 to 23.8%), hybridizing with LAC435 and RUM831, respectively; and the classes Deltaproteobacteria (5.8 to 28.3%) and Gammaproteobacteria (1.2 to 8.2%). All were more abundant in the rumen communities of cows fed diets containing silage (75.2 to 87.3%) than in those of cows fed diets containing hay (31.8 to 49.5%). The addition of flaxseed reduced their abundance in the rumens of cows fed silage-based diets (to 45.2 to 58.7%) but did not change markedly their abundance in the rumens of cows fed hay-based diets (31.8 to 49.5%). Fibrolytic species, including Fibrobacter succinogenes and Ruminococcus spp., and archaeal methanogens accounted for only a small proportion (0.4 to 2.1% and 0.2 to 0.6%, respectively) of total cell numbers. Depending on diet, between 37.0 and 91.6% of microbial cells specifically hybridized with the probes used in this study, allowing them to be identified in situ. The identities of other microbial populations (8.4 to 63.0%) remain unknown.

Combined administration of ghrelin and GHRH synergistically stimulates GH release in Holstein preweaning calves.

Ghrelin is a gut peptide which participates in growth regulation through its somatotropic, lipogenic and orexigenic effects. Synergism of ghrelin and growth hormone-releasing hormone (GHRH) on growth hormone (GH) secretion has been reported in humans and rats, but not in domestic animals in vivo. In this study, effects of a combination of ghrelin and GHRH on plasma GH and other metabolic parameters, and changes in plasma active and total ghrelin levels were studied in Holstein bull calves before and after weaning. Six calves were intravenously injected with vehicle (0.1% BSA-saline), ghrelin (1 microg/kg BW), GHRH (0.25 microg/kg BW) or a combination of ghrelin plus GHRH at the age of 5 weeks and 10 weeks (weaning at 6 weeks of age). Ghrelin stimulated GH release with similar potency as GHRH and their combined administration synergistically stimulated GH release in preweaning calves. After weaning, GH responses to ghrelin and GHRH became greater compared with the values of preweaning calves, but a synergistic effect of ghrelin and GHRH was not observed. The GH areas under the concentration curves for 2h post-injection were greater in weaned than in preweaning calves (P<0.05) if ghrelin or GHRH were injected alone, but were similar if ghrelin and GHRH were injected together. Basal plasma active and total ghrelin levels did not change around weaning, but transiently increased after ghrelin injection. Basal plasma insulin, glucose and non-esterified fatty acid levels were reduced after weaning, but no changes by treatments were observed. In conclusion, ghrelin and GHRH synergistically stimulated GH release in preweaning calves, but this effect was lost after weaning.