Supplementation with rumen-inert fat in the growing phase altered adipogenic gene expression and the size and number of adipocytes in Hanwoo steers.

We hypothesized that the provision of rumen-inert fat (RIF) to growing cattle (9 to 13 mo of age) would affect the expression of genes involved in lipid metabolism and thereby affect the size and number of adipocytes of steers slaughtered at 30 mo of age. Thirty steers with an average initial body weight (BW) of 239 ±â€…25 kg were allocated to six pens, balanced for BW and genetic merit for marbling, and assigned to one of two treatment groups: control (only basal diet) or test diet (basal diet with 200 g of RIF per day, on an as-fed basis) for 5 mo. Biopsy samples of longissimus lumborum (LM) muscle were then collected for analysis of fatty acid composition and gene expression. Both groups were then fed the same basal diets during the early and late fattening phases, without RIF, until slaughter (average shrunk BW = 759 kg). Supplementation with RIF increased the longissimus thoracis (LT) intramuscular fatty acid concentration at slaughter (P = 0.087) and numerically increased the quality grade score (P = 0.106). The LM intramuscular relative mRNA expression of genes such as PPARα, ZFP423 and SREBP1, FASN, SCD, FABP4, GPAT1, and DGAT2 were downregulated (P < 0.1) following RIF supplementation. Supplementation of RIF decreased (P < 0.1) diameter and concomitantly increased intramuscular adipocytes per viewing section at slaughter. This likely was caused by promotion of triacylglycerol hydrolysis during the growing phase. Another possible explanation is that the relative mRNA expression of gene ATGL was upregulated by RIF supplementation during the growing (P < 0.1) and the fattening phases (P < 0.05), while the genes associated with fatty acid uptake (FABP4) and esterification (DGAT2) were downregulated during the growing phase and upregulated (P < 0.1) during the fattening phase. This implies that the lipid turnover rate was higher for steers during the growing than fattening phase. This study demonstrated that RIF supplementation during the growing phase induced a carryover effect on the lipogenic transcriptional regulation involved in adipocyte lipid content of intramuscular adipose tissue; increased triacylglycerol hydrolysis during the growing phase subsequently was followed by increased lipid accumulation during the fattening phases.

Rumen inert fat (RIF) is a type of fat supplement that is used in the diets of beef cattle as early as 6 mo of age in calves and continues through the finishing period to improve the dietary energy density which can be used by the animal to deposit more lipid in the muscle tissue. However, for Hanwoo beef cattle, the precise time of RIF supplementation has not yet been determined. This study hypothesized that supplementing RIF at the growing phase (9 to 13 mo of age) would have a positive influence on the marbling characteristics of meat at slaughter. The growth rate and performance of steers were not improved by RIF supplementation, however, an increase in intramuscular fatty acid content was noted that was accompanied by the increased number of intramuscular adipocytes and decreased intramuscular adipocyte diameter. Supportively, upregulation of the genes associated with fatty acid uptake and esterification during the fattening phase of RIF-fed animals was noted. Overall, supplementing RIF at the growing stage could improve the lipid content of the meat which is supported by the increased lipid hydrolysis during the growing phase and followed by increased lipid accumulation during the fattening phases.

Metabolite Profile, Ruminal Methane Reduction, and Microbiome Modulating Potential of Seeds of Pharbitis nil.

We identified metabolites in the seeds of Pharbitis nil (PA) and evaluated their effects on rumen methanogenesis, fiber digestibility, and the rumen microbiome in vitro and in sacco. Four rumen-cannulated Holstein steers (mean body weight 507 ± 32 kg) were used as inoculum donor for in vitro trial and live continuous culture system for in sacco trial. PA was tested in vitro at doses ranging from 4.5 to 45.2% dry matter (DM) substrate. The in sacco trial was divided into three phases: a control phase of 10 days without nylon bags containing PA in the rumen, a treatment phase of 11 days in which nylon bags containing PA (180 g) were placed in the rumen, and a recovery phase of 10 days after removing the PA-containing bags from the rumen. Rumen headspace gas and rumen fluid samples were collected directly from the rumen. PA is enriched in polyunsaturated fatty acids dominated by linoleic acid (C18:2) and flavonoids such as chlorogenate, quercetin, quercetin-3-O-glucoside, and quinic acid derivatives. PA decreased (p < 0.001) methane (CH4) production linearly in vitro with a reduction of 24% at doses as low as 4.5% DM substrate. A quadratic increase (p = 0.078) in neutral detergent fiber digestibility was also noted, demonstrating that doses < 9% DM were optimal for simultaneously enhancing digestibility and CH4 reduction. In sacco, a 50% decrease (p = 0.087) in CH4 coupled with an increase in propionate suggested increased biohydrogenation in the treatment phase. A decrease (p < 0.005) in ruminal ammonia nitrogen (NH3-N) was also noted with PA in the rumen. Analysis of the rumen microbiome revealed a decrease (p < 0.001) in the Bacteroidetes-to-Firmicutes ratio, suggesting PA to have antiprotozoal potential. At the genus level, a 78% decrease in Prevotella spp. and a moderate increase in fibrolytic Ruminococcus spp. were noted in the treatment phase. In silico binding of PA metabolites to cyclic GMP-dependent protein kinase of Entodinium caudatum supported the antiprotozoal effect of PA. Overall, based on its high nutrient value and antiprotozoal activity, PA could probably replace the ionophores used for CH4 abatement in the livestock industry.

Effects of Dietary Protein Concentration on Lipid Metabolism Gene Expression and Fatty Acid Composition in 18-23-Month-Old Hanwoo Steers.

The present study evaluated the influence of dietary protein level on growth performance, fatty acid composition, and the expression of lipid metabolic genes in intramuscular adipose tissues from 18- to 23-month-old Hanwoo steers, representing the switching point of the lean-to-fat ratio. Forty steers with an initial live weight of 486 ± 37 kg were assigned to one of two treatment groups fed either a concentrate diet with 14.5% CP and or with 17% CP for 6 months. Biopsy samples of intramuscular tissue were collected to analyze the fatty acid composition and gene expression at 23 months of age. Throughout the entire experimental period, all steers were restrained twice daily to allow individual feeding. Growth performance, blood metabolites, and carcass traits, according to ultrasonic measurements, were not affected by the experimental diets. The high-protein diet significantly increased the expression of intramuscular PPARα (p < 0.1) and LPL (p < 0.05) but did not affect genes involved in fatty acid uptake (CD36 and FABP4) nor lipogenesis (ACACA, FASN, and SCD). In addition, it downregulated intramuscular VLCAD (p < 0.01) related to lipogenesis but also GPAT1 (p = 0.001), DGAT2 (p = 0.016), and SNAP23 (p = 0.057), which are involved in fatty acid esterification and adipocyte size. Hanwoo steers fed a high-protein diet at 18-23 months of age resulted in a relatively lower lipid turnover rate than steers fed a low-protein diet, which could be responsible for shortening the feeding period.

Feeding Systems and Host Breeds Influence Ruminal Fermentation, Methane Production, Microbial Diversity and Metagenomic Gene Abundance.

Our previous research revealed the advantages of separate feeding (SF) systems compared to total mixed ration (TMR) in terms of ruminal methane (CH4) production. The purpose of this experiment was to confirm the advantage of SF as a nutritional strategy for CH4 mitigation, and to determine the effects of different feeding systems (TMR and SF) on the rumen microbiome and associated metagenome of two different breeds and on CH4 emissions. We randomly allocated four Holstein (305 ± 29 kg) and four Hanwoo steers (292 ± 24 kg) to two groups; the steers were fed a commercial concentrate with tall fescue (75:25) as TMR or SF, in a crossover design (two successive 22-day periods). Neither feeding systems nor cattle breeds had an effect on the total tract digestibility of nutrients. The TMR feeding system and Hanwoo steers generated significantly more CH4 (P < 0.05) and had a higher yield [g/d and g/kg dry matter intake (DMI)] compared to the SF system and Holstein steers. A larger rumen acetate:propionate ratio was observed for the TMR than the SF diet (P < 0.05), and for Hanwoo than Holstein steers (P < 0.001), clearly reflecting a shift in the ruminal H2 sink toward CH4 production. The linear discriminant analysis (LDA) effect size (LEfSe) revealed a greater abundance (α < 0.05 and LDA > 2.0) of operational taxonomic units (OTUs) related to methanogenesis for Hanwoo steers compared to Holstein steers. Kendall's correlation analysis revealed wide variation of microbial co-occurrence patterns between feeding systems, indicating differential H2 thermodynamics in the rumen. A metagenome analysis of rumen microbes revealed the presence of 430 differentially expressed genes, among which 17 and 27 genes exhibited positive and negative associations with CH4 production, respectively (P < 0.001). A strong interaction between feeding system and breed was observed for microbial and metagenomic abundance. Overall, these results suggest that the TMR feeding system produces more CH4, and that Hanwoo cattle are higher CH4 emitters than SF diet and Holstein cattle, respectively. Interestingly, host-associated microbial interactions differed within each breed depending on the feeding system, which indicated that breed-specific feeding systems should be taken into account for farm management.

Developing Equations for Converting Digestible Energy to Metabolizable Energy for Korean Hanwoo Beef Cattle.

This study was performed to update and generate prediction equations for converting digestible energy (DE) to metabolizable energy (ME) for Korean Hanwoo beef cattle, taking into consideration the gender (male and female) and body weights (BW above and below 350 kg) of the animals. The data consisted of 141 measurements from respiratory chambers with a wide range of diets and energy intake levels. A simple linear regression of the overall unadjusted data suggested a strong relationship between the DE and ME (Mcal/kg DM): ME = 0.8722 × DE + 0.0016 (coefficient of determination (R2) = 0.946, root mean square error (RMSE) = 0.107, p < 0.001 for intercept and slope). Mixed-model regression analyses to adjust for the effects of the experiment from which the data were obtained similarly showed a strong linear relationship between the DE and ME (Mcal/kg of DM): ME = 0.9215 × DE - 0.1434 (R2 = 0.999, RMSE = 0.004, p < 0.001 for the intercept and slope). The DE was strongly related to the ME for both genders: ME = 0.8621 × DE + 0.0808 (R2 = 0.9600, RMSE = 0.083, p < 0.001 for the intercept and slope) and ME = 0.7785 × DE + 0.1546 (R2 = 0.971, RMSE = 0.070, p < 0.001 for the intercept and slope) for male and female Hanwoo cattle, respectively. By BW, the simple linear regression similarly showed a strong relationship between the DE and ME for Hanwoo above and below 350 kg BW: ME = 0.9833 × DE - 0.2760 (R2 = 0.991, RMSE = 0.055, p < 0.001 for the intercept and slope) and ME = 0.72975 × DE + 0.38744 (R2 = 0.913, RMSE = 0.100, p < 0.001 for the intercept and slope), respectively. A multiple regression using the DE and dietary factors as independent variables did not improve the accuracy of the ME prediction (ME = 1.149 × DE - 0.045 × crude protein + 0.011 × neutral detergent fibre - 0.027 × acid detergent fibre + 0.683).

In Vitro Screening of East Asian Plant Extracts for Potential Use in Reducing Ruminal Methane Production.

Indiscriminate use of antibiotics can result in antibiotic residues in animal products; thus, plant compounds may be better alternative sources for mitigating methane (CH4) production. An in vitro screening experiment was conducted to evaluate the potential application of 152 dry methanolic or ethanolic extracts from 137 plant species distributed in East Asian countries as anti-methanogenic additives in ruminant feed. The experimental material consisted of 200 mg total mixed ration, 20 mg plant extract, and 30 mL diluted ruminal fluid-buffer mixture in 60 mL serum bottles that were sealed with rubber stoppers and incubated at 39 °C for 24 h. Among the tested extracts, eight extracts decreased CH4 production by >20%, compared to the corresponding controls: stems of Vitex negundo var. incisa, stems of Amelanchier asiatica, fruit of Reynoutria sachalinensis, seeds of Tribulus terrestris, seeds of Pharbitis nil, leaves of Alnus japonica, stem and bark of Carpinus tschonoskii, and stems of Acer truncatum. A confirmation assay of the eight plant extracts at a dosage of 10 mg with four replications repeated on 3 different days revealed that the extracts decreased CH4 concentration in the total gas (7-15%) and total CH4 production (17-37%), compared to the control. This is the first report to identify the anti-methanogenic activities of eight potential plant extracts. All extracts decreased ammonia (NH3-N) concentrations. Negative effects on total gas and volatile fatty acid (VFA) production were also noted for all extracts that were rich in hydrolysable tannins and total saponins or fatty acids. The underlying modes of action differed among plants: extracts from P. nil, V. negundo var. incisa, A. asiatica, and R. sachalinensis resulted in a decrease in total methanogen or the protozoan population (p < 0.05) but extracts from other plants did not. Furthermore, extracts from P. nil decreased the population of total protozoa and increased the proportion of propionate among VFAs (p < 0.05). Identifying bioactive compounds in seeds of P. nil by gas chromatography-mass spectrometry analysis revealed enrichment of linoleic acid (18:2). Overall, seeds of P. nil could be a possible alternative to ionophores or oil seeds to mitigate ruminal CH4 production.

Inhibitory potential of EGCG on Streptococcus mutans biofilm: A new approach to prevent Cariogenesis.

Dental caries is a common cause for tooth loss and Streptococcus mutans is identified as the etiologic pathogen. This study evaluates the inhibitory potential of Epigallocatechin gallate (EGCG) on S.mutans glucansucrase enzyme and its biofilm. Glucansucrase binding and the inhibitory potential of EGCG was validated using AutoDock tool and enzyme inhibitory assay. Biofilm inhibitory potential was also confirmed using Scanning Electron Microscopic (SEM) analysis in human tooth samples. Molecular docking revealed that EGCG interacted with GLU 515 and TRP 517 amino acids and binds to glucansucrase. SEM analysis revealed inhibition of S.mutans biofilm by various concentrations of EGCG on surfaces of tooth samples. Bioinformatics and biological assays confirmed that EGCG potentially binds to the S. mutans glucansucrase and inhibits its enzymatic activity. Enzymatic inhibition of glucansucrase attenuated biofilm formation potential of S. mutans on tooth surface. Thus, we conclude that EGCG inhibitory potential of S. mutans biofilm on the tooth surface is a novel approach in prevention of dental caries.

Chemical composition, antioxidant activity and antibacterial mechanism of action from Marsilea minuta leaf hexane: methanol extract.

BACKGROUND: In the present study, hexane: methanol (50:50) leaf extract of Marisela minuta has been evaluated for its chemical composition, antioxidant effect and the antimicrobial mechanism of action against food borne pathogenic bacteria. RESULTS: The phytochemical evaluation of extract by GC/MS revealed the major abundance of benzoic acid-4-ethoxyethyl ester (43.39%) and farnesol acetate (18.42%). The extract exhibited potential antioxidant and free radical scavenging properties with promising antibacterial activities against the test pathogens with Pseudomonas aeruginosa being the most susceptible with maximum inhibition zone (17 mm) and IC50 value of 125 µg, respectively. The significant (p < 0.05) increase in intracellular super oxide dismutase (SOD), protein leakage, extracellular alkaline phosphatase and lactate dehydrogenase in treated test pathogens suggested an increase in oxidative stress reveling the mechanism of action of phytochemicals. Scanning electron microscopy analysis of treated pathogens also showed swollen and distorted cells. The bioactive molecules in the extract were efficiently docked with virulent enzymes and farnesol acetate showed best energy value of - 5.19 and - 4.27 kcal/mol towards Topoisomerase IV and SHV-2 respectively. Benzoic acid-4-ethoxyethyl ester showed best binding against TEM-72 with low binding energy value of - 4.35 kcal/mol. CONCLUSION: Due to its antioxidant and antibacterial properties, the leaf extract of M. minuta may act as promising natural additives to prevent food spoilage bacteria.

Ruminal methane emissions, metabolic, and microbial profile of Holstein steers fed forage and concentrate, separately or as a total mixed ration.

Few studies have examined the effects of feeding total mixed ration (TMR) versus roughage and concentrate separately (SF) on ruminant methane production. Therefore, this study compared differences in methane production, ruminal characteristics, total tract digestibility of nutrients, and rumen microbiome between the two feeding methods in Holstein steers. A total six Holstein steers of initial bodyweights 540 ± 34 kg were divided into two groups and assigned to a same experimental diet with two different feeding systems (TMR or SF) in a crossover design with 21 d periods. The experimental diet contained 73% concentrate and 27% forage and were fed twice a day. The total tract digestibility of crude protein, neutral detergent fibre, and organic matter were not affected by the two different feeding systems. Steers fed TMR emitted more methane (138.5 vs. 118.2 L/d; P < 0.05) and lost more gross energy as methane energy (4.0 vs. 3.5% gross energy intake; P = 0.005) compared to those fed SF. Steers fed TMR had greater (P < 0.05) total volatile fatty acid (VFA), ammonia-N concentrations and propionate proportion of total VFA at 1.5 h, whereas lower after that compared to steers fed SF. The greater (P < 0.05) acetate: propionate ratio at 4.5 h for steers fed TMR reflected the shift of H2 sink from propionate towards acetate synthesis. The lower (P < 0.05) isobutyrate and isovalerate proportions of total VFA observed in steers fed TMR implies decrease in net consumption of H2 for microbial protein synthesis compared to SF. There were no differences in both major bacterial and archaeal diversity between TMR and SF, unlike several minor bacterial abundances. The minor groups such as Coprococcus, Succiniclasticum, Butyrivibrio, and Succinivibrio were associated with the changes in ruminal VFA profiles or methanogenesis indirectly. Overall, these results indicate that SF reduces methane emissions from ruminants and increases propionate proportion of total VFA without affecting total tract digestion compared to TMR. There were no evidences that the response differed due to different major underlying microbial population.

Identification of evolutionarily conserved Momordica charantia microRNAs using computational approach and its utility in phylogeny analysis.

Momordica charantia (bitter gourd, bitter melon) is a monoecious Cucurbitaceae with anti-oxidant, anti-microbial, anti-viral and anti-diabetic potential. Molecular studies on this economically valuable plant are very essential to understand its phylogeny and evolution. MicroRNAs (miRNAs) are conserved, small, non-coding RNA with ability to regulate gene expression by bind the 3' UTR region of target mRNA and are evolved at different rates in different plant species. In this study we have utilized homology based computational approach and identified 27 mature miRNAs for the first time from this bio-medically important plant. The phylogenetic tree developed from binary data derived from the data on presence/absence of the identified miRNAs were noticed to be uncertain and biased. Most of the identified miRNAs were highly conserved among the plant species and sequence based phylogeny analysis of miRNAs resolved the above difficulties in phylogeny approach using miRNA. Predicted gene targets of the identified miRNAs revealed their importance in regulation of plant developmental process. Reported miRNAs held sequence conservation in mature miRNAs and the detailed phylogeny analysis of pre-miRNA sequences revealed genus specific segregation of clusters.

Characterization of ambrette seed oil and its mode of action in bacteria.

In the present study, chemical composition and the antibacterial mechanism of ambrette seed oil are investigated. Chemical composition of the oil was analysed by gas chromatography-mass spectrometry (GC-MS). Thirty-five compounds were identified and the major compounds were found to be farnesol acetate (51.45%) and ambrettolide (12.96%). The antibacterial activity was performed by well diffusion assay and the mechanisms were studied by measuring the alkaline phosphatase (ALP), lactate dehydrogenase (LDH) and protein leakage assays. The antibacterial effect of the ambrette seed oil showed inhibitory effect against Bacillus subtilis, Staphylococcus aureus and Enterococcus faecalis. The LDH activity was high in all tested bacteria compared with control, whereas the ALP and protein concentrations were also increased in E. faecalis. Molecular docking revealed the ligands farnesol acetate and ambrettolide had satisfactory binding energy towards the beta lactamase TEM-72 and dihydrofolate reductase (DHFR) protein. Due to its better antibacterial properties, the ambrette seed oil could be used as a source of antibacterial agents.