A.P. Faciola

Camelina Seed Supplementation at Two Dietary Fat Levels Change Ruminal Bacterial Community Composition in a Dual-Flow Continuous Culture System

This experiment aimed to determine the effects of camelina seed (CS) supplementation at different dietary fat levels on ruminal bacterial community composition and how it relates to changes in ruminal fermentation in a dual-flow continuous culture system. Diets were randomly assigned to 8 fermenters (1,200–1,250 mL) in a 2 × 2 factorial arrangement of treatments in a replicated 4 × 4 Latin square with four 10-day experimental periods that consisted of 7 days for diet adaptation and 3 days for sample collection. Treatments were: (1) no CS at 5% ether extract (EE, NCS5); (2) no CS at 8% EE (NCS8); (3) 7.7% CS at 5% EE (CS5); and (4) 17.7% CS at 8% EE (CS8). Megalac was used as a control to adjust EE levels. Diets contained 55% orchardgrass hay and 45% concentrate, and fermenters were equally fed a total of 72 g/day (DM basis) twice daily. The bacterial community was determined by sequencing the V4 region of the 16S rRNA gene using the Illumina MiSeq platform. Sequencing data were analyzed using mothur and statistical analyses were performed in R and SAS. The most abundant phyla across treatments were the Bacteroidetes and Firmicutes, accounting for 49 and 39% of the total sequences, respectively. The bacterial community composition in both liquid and solid fractions of the effluent digesta changed with CS supplementation but not by dietary EE. Including CS in the diets decreased the relative abundances of Ruminococcus spp., Fibrobacter spp., and Butyrivibrio spp. The most abundant genus across treatments, Prevotella, was reduced by high dietary EE levels, while Megasphaera and Succinivibrio were increased by CS supplementation in the liquid fraction. Correlatively, the concentration of acetate was decreased while propionate increased; C18:0 was decreased and polyunsaturated fatty acids, especially C18:2 n-6 and C18:3 n-3, were increased by CS supplementation. Based on the correlation analysis between genera and fermentation end products, this study revealed that CS supplementation could be energetically beneficial to dairy cows by increasing propionate-producing bacteria and suppressing ruminal bacteria associated with biohydrogenation. However, attention should be given to avoid the effects of CS supplementation on suppressing cellulolytic bacteria.

Effects of methionine plus cysteine inclusion on performance and body composition of liquid-fed crossbred calves fed a commercial milk replacer and no starter feed

This experiment aimed to evaluate the effects of supplying 4 different inclusion levels of Met + Cys to crossbred liquid-fed calves on animal performance and body composition. Thirty-six Holstein-Gyr male calves were separated into 2 age groups: 16 calves, slaughtered at an age of 30 d, representing the physiological phase from 8 to 30 d, and 20 calves, slaughtered at an age of 60 d, representing the physiological phase from 30 to 60 d. At 8 d of age, the animals were randomly distributed among the experimental treatments: 4 Met + Cys inclusion levels (Met + Cys: 8.0, 8.7, 9.4, and 10.2 g/d), provided by an AA supplement added to 1.0 kg (as fed) of commercial milk replacer containing soy protein concentrate and wheat protein isolate reconstituted at 13.8% (dry matter basis). The diet was supplied without allowing leftovers and no starter feed was provided. The experimental diets were supplied without allowing orts, so that the dry matter, crude protein, and ether extract intakes were the same for all animals, independent of Met + Cys level. Total weight gain, average daily gain, gain composition, and body composition were evaluated for both age groups separately. Digestibility of organic matter, crude protein, and ether extract was lower for 8 to 30 d than for 30 to 60 d. The effect of Met + Cys levels on the digestibility of nutrients was not observed; there also was no significant interaction between physiological phase and Met + Cys levels. For the 8 to 30 d group, no responses in performance were observed according to the different Met + Cys levels, which indicates that 8.0 g/d of Met + Cys met the requirements for this physiological phase. The 30 to 60 d group responded positively to higher Met + Cys inclusion in the diet. In conclusion, an optimal Met + Cys dietary level to ensure best performance and protein gain ranges from 8.41 to 9.81 g/d.

Effect of replacing calcium salts of palm oil with camelina seed at 2 dietary ether extract levels on digestion, ruminal fermentation, and nutrient flow in a dual-flow continuous culture system

Camelina is a drought- and salt-tolerant oil seed, which in total ether extract (EE) contains up to 74% polyunsaturated fatty acids. The objective of this study was to assess the effects of replacing calcium salts of palm oil (Megalac, Church & Dwight Co. Inc., Princeton, NJ) with camelina seed (CS) on ruminal fermentation, digestion, and flows of fatty acids (FA) and AA in a dual-flow continuous culture system when supplemented at 5 or 8% dietary EE. Diets were randomly assigned to 8 fermentors in a 2 × 2 factorial arrangement of treatments in a replicated 4 × 4 Latin square design, with four 10-d experimental periods consisting of 7 d for diet adaptation and 3 d for sample collection. Treatments were (1) calcium salts of palm oil supplementation at 5% EE (MEG5); (2) calcium salts of palm oil supplementation at 8% EE (MEG8); (3) 7.7% CS supplementation at 5% EE (CS5); and (4) 17.7% CS supplementation at 8% EE (CS8). Diets contained 55% orchardgrass hay, and fermentors were fed 72 g of dry matter/d. On d 8, 9, and 10 of each period, digesta effluent samples were taken for ruminal NH3, volatile fatty acids, nitrogen metabolism analysis, and long-chain FA and AA flows. Statistical analysis was performed using the MIXED procedure (SAS Institute Inc., Cary, NC). We detected an interaction between FA source and dietary EE level for acetate, where MEG8 had the greatest molar proportion of acetate. Molar proportions of propionate were greater and total volatile fatty acids were lower on CS diets. Supplementation of CS decreased overall ruminal nutrient true digestibility, but dietary EE level did not affect it. Diets containing CS had greater biohydrogenation of 18:2 and 18:3; however, biohydrogenation of 18:1 was greater in MEG diets. Additionally, CS diets had greater ruminal concentrations of trans-10/11 18:1 and cis-9,trans-11 conjugated linoleic acid. Dietary EE level at 8% negatively affected flows of NH3-N (g/d), nonammonia N, and bacterial N as well as the overall AA outflow. However, treatments had minor effects on individual ruminal AA digestibility. The shift from acetate to propionate observed on diets containing CS may be advantageous from an energetic standpoint. Moreover, CS diets had greater ruminal outflow of trans-10/11 18:1 and cis-9,trans-11 conjugated linoleic acid than MEG diets, suggesting a better FA profile available for postruminal absorption. However, dietary EE at 8% was deleterious to overall N metabolism and AA outflow, indicating that CS can be fed at 5% EE without compromising N metabolism.

Rapid Communication: Prolactin and hydrocortisone impact TNFα-mediated mitogen-activated protein kinase signaling and inflammation of bovine mammary epithelial (MAC-T) cells1

The objective of this study was to evaluate the effects of the hormones prolactin (PRL) and hydrocortisone (HC) on bovine mammary alveolar (MAC-T) cells mitogen-activated protein kinase (MAPK) inflammatory signaling and inflammatory gene expression. MAC-T cells were cultured in the presence (+PRL +HC; Dulbeccos modified Eagles medium [DMEM] 10% fetal bovine serum, 10 µg/mL of insulin, 100 IU/mL penicillin, 100 µg/mL streptomycin, 1 µg/mL ovine PRL, 0.5 µg/mL HC, and 10 m sodium acetate) or the absence (-PRL -HC; DMEM 10% fetal bovine serum, 10 µg/mL insulin, 100 IU/mL penicillin , and 100 µg/mL streptomycin) of PRL and HC, and MAPK (extracellular signal-regulated kinase [ERK], c-Jun N-terminal kinase [JNK], and p38) phosphorylation and inflammatory gene expression were examined in response to tumor necrosis factor α (TNFα). Statistical analysis was assessed using 1-way ANOVA, and Tukeys post hoc analysis was used to assess statistical significance when ≤ 0.05. MAC-T cells cultured in +PRL +HC and -PRL -HC were co-stimulated with increasing concentrations of TNFα (0, 10, 30, 100, 300, and 1,000 p). Cell lysates were harvested 15 min after TNFα stimulation and assessed for MAPK phosphorylation using immunoblotting. c-Jun N-terminal kinase and p38 phosphorylation increased in a dose-dependent manner and was greater in cells cultured in -PRL -HC. MAC-T cells cultured in +PRL +HC and -PRL -HC were next stimulated with TNFα (300 p), and lysates were harvested over time (0, 15, 30, 60, 120, and 180 min) after TNFα stimulation. c-Jun N-terminal kinase and p38 phosphorylation was transiently increased in MAC-T cells stimulated with TNFα; however, JNK and p38 signaling was greater in MAC-T cells cultured in -PRL -HC. We next examined inflammatory gene expression in MAC-T cells cultured in +PRL +HC and -PRL -HC. Cells were co-stimulated with (300 p) or without TNFα. Ribonucleic acid was isolated 1 h after TNFα stimulation, and a PCR array was performed to examine the expression of 83 inflammatory genes. Gene expression was increased in MAC-T cells in response to TNFα. Consistent with enhanced MAPK signaling, inflammatory gene expression was increased in MAC-T cells cultured in -PRL -HC. Real-time quantitative PCR of 6 target genes was used to validate the PCR array findings. Collectively, our data demonstrate that -PRL -HC MAC-T cells are more responsive to TNFα stimuli. These findings suggest that cell culture conditions (e.g., treatment with hormones) greatly impact cellular response and should be considered prior to experimental design and hypothesis testing.

Does partial replacement of corn with glycerin in beef cattle diets affect in vitro ruminal fermentation, gas production kinetic, and enteric greenhouse gas emissions?

Five in vitro experiments were conducted with the following objectives: 1) To evaluate the ruminal fermentation of three different single ingredients: corn, glycerin, and starch (Exp. 1 and 2); 2) To assess effects of partially replacing corn with glycerin in beef cattle diets on ruminal fermentation pattern (Exp. 3 and 4); and 3) To evaluate the effects of glycerin inclusion on the extension of ruminal DM digestibility of feeds with high (orchard hay) and low (corn) fiber content (Exp. 5). For Exp. 1 and 2, two in vitro systems (24-bottle AnkomRF and 20-serum bottles) were used in four consecutive fermentation batches to evaluate gas production (GP), fermentation profiles, enteric methane (CH4), and carbon dioxide (CO2) of corn, glycerin, and starch. The 24 h total GP, acetate concentration, and acetate: propionate ratio decreased only when glycerin was added to the diet (P < 0.01). The 48-h total GP and metabolizable energy were greatest for corn (P < 0.01), and similar between glycerin and starch. The starch treatment had the lowest total volatile fatty acids concentration (P = 0.01). Glycerin had greatest CH4 production, lag time, and maximum gas volume of the first pool (P < 0.05). However, the maximum gas volume of the second pool was greatest for corn (P < 0.05), and similar between glycerin and starch. The starch treatment had the greatest specific rates of digestion for first and second pools (P < 0.05). Production of CO2 (mL/g) was greater for corn (P < 0.01), but similar for glycerin and starch. For Exp. 3 and 4, the same systems were used to evaluate four different levels of glycerin [0, 100, 200, and 300 g/kg of dry matter (DM)] replacing corn in beef cattle finishing diets. Glycerin levels did not affect 24 and 48 h total GP, CH4, and CO2 (P > 0.05). The inclusion of glycerin linearly decreased acetate concentration (P = 0.03) and acetate: propionate ratio (P = 0.04). For Exp. 5, two DaisyII incubators were used to evaluate the in vitro dry matter digestibility (IVDMD) of the following treatments: orchard hay; corn; orchard hay + glycerin; and corn + glycerin. Glycerin inclusion decreased orchard hay IVDMD (P < 0.01) but did not affect corn IVDMD (P > 0.05). We concluded that, under these experimental conditions, glycerin has similar energy efficiency when used in replacement of corn and included at up to 300 g/kg in beef cattle diets.

Effects of lipopolysaccharide dosing on bacterial community composition and fermentation in a dual-flow continuous culture system

The objectives of this study were to evaluate the effects of lipopolysaccharide (LPS) dosing on bacterial fermentation and bacterial community composition (BCC), to set up a subacute ruminal acidosis (SARA) nutritional model in vitro, and to determine the best sampling time for LPS dosing in a dual-flow continuous culture system. Diets were randomly assigned to 6 fermentors in a replicated 3 × 3 Latin square with three 11-d experimental periods that consisted of 7 d for diet adaptation and 4 d for sample collection. Treatments were control diet (CON), wheat and barley diet (WBD) to induce SARA, and control diet + LPS (LPSD). Fermenters were fed 72 g of dry matter/d. The forage:concentrate ratio of CON was 65:35. The WBD diet was achieved by replacing 40% of dry matter of the CON diet with 50% ground wheat and 50% ground barley. The LPS concentration in LPSD was 200,000 endotoxin units, which was similar to that observed in cows with SARA. The SARA inducing and LPS dosing started at d 8. The BCC was determined by sequencing the V4 region of the 16S rRNA gene using the Illumina MiSeq platform (Illumina Inc., San Diego, CA). The LPSD and CON maintained pH above 6 for the entire experimental period, and the WBD kept pH between 5.2 and 5.6 for 4 h/d, successfully inducing SARA. Digestibility of neutral detergent fiber and crude protein in LPSD were not different from WBD but tended to be lower than CON. Lipopolysaccharide dosing had no effect on pool of VFA concentrations and profiles but decreased bacterial N; the pattern changes of VFA and LPS in LPSD started to increase and be similar to WBD 6 h after LPS dosing. Pool of LPS concentration was around 11-fold higher in WBD and 4-fold higher in LPSD than CON. In the solid fraction, the BCC of LPSD was different from WBD and tended to be different from CON. In the liquid fraction, the BCC was different among treatments. The LPS dosing increased the relative abundance of Succinimonas, Anaeroplasma, Succinivibrio, Succiniclasticum, and Ruminobacter, which are main gram-negative bacteria related to starch digestion. Our results suggest that LPS dosing does not affect pH alone. However, LPS could drive the development of SARA by affecting bacteria and bacterial fermentation. For future studies, samples are suggested to be taken 6 h after LPS dosing in a dual-flow continuous culture system.

Effects of replacing canola meal with solvent-extracted camelina meal on microbial fermentation in a dual-flow continuous culture system

Camelina is an oil seed crop that belongs to the Brassica family (Cruciferae). Camelina meal is a by-product from the biofuel industry that contains on average 38% crude protein and between 10 to 20% of residual fat, which limits the inclusion levels of camelina meal in dairy cow diets as the main protein supplement. Thus, we conducted a solvent extraction on ground camelina seed on a laboratory scale. The objectives of this study were (1) to assess the effects of replacing canola meal (CM) with solvent-extracted camelina meal (SCAM) in lactating dairy cow diets; and (2) to determine the effects of SCAM on microbial fermentation and AA flow in a dual-flow continuous culture system. Diets were randomly assigned to 6 fermentors in a replicated 3 × 3 Latin square with three 10-d experimental periods consisting of 7 d for diet adaptation and 3 d for sample collection. Treatments were 0, 50, and 100% SCAM inclusion, replacing CM as the protein supplement. Diets contained 55:45 forage:concentrate, and fermentors were fed 72 g of dry matter/d equally divided in 2 feeding times. On d 8, 9, and 10 of each period, samples were collected for analyses of pH, volatile fatty acids (VFA), N metabolism, NH3-N, digestibility, and AA flow. Statistical analysis was performed using the MIXED procedure of SAS (SAS Institute Inc., Cary, NC), and linear and quadratic effects of SCAM inclusion were assessed. Total VFA concentration and pH were not affected by diets. Molar proportion of acetate decreased, whereas molar proportion of propionate increased with SCAM inclusion. Total branched-chain VFA concentration was the least in fermentors fed diet 0, and greatest in fermentors fed diet 50. Digestibility of NDF decreased in fermentors fed SCAM diets, and dry matter, organic matter, and crude protein true digestibility were similar across diets. Concentration of NH3-N linearly decreased, and non-NH3-N linearly increased with SCAM inclusion. Bacterial efficiency (calculated as g of bacterial N flow/kg of organic matter truly digested) tended to be greater in fermentors fed diet 100. Outflow of Arg linearly increased with SCAM inclusion, whereas overall AA flow was not affected by diet. In conclusion, replacing CM with SCAM increased propionate molar proportion and non-NH3-N flow, and decreased NH3-N flow and concentration, which may improve animal energy status and N utilization. Inclusion of SCAM did not change most AA flow, indicating that it can be a potential replacement for CM.

HDAC1/2-mediated regulation of JNK and ERK phosphorylation in bovine mammary epithelial cells in response to TNF-α: ROMANICK et al.

Bovine mammary epithelial cells (MAC‐Ts) are a common cell line for the study of mammary epithelial inflammation; these cells are used to mechanistically elucidate molecular underpinnings that contribute to bovine mastitis. Bovine mastitis is the most prevalent form of disease in dairy cattle that culminates in annual losses of two billion dollars for the US dairy industry. Thus, there is an urgent need for improved therapeutic strategies. Histone deacetylase (HDAC) inhibitors are efficacious in rodent models of inflammation, yet their role in bovine mammary cells remain unclear. HDACs have traditionally been studied in the regulation of nucleosomal DNA, in which deacetylation of histones impact chromatin accessibility and gene expression. Using MAC‐T cells stimulated with tumor necrosis factor α (TNF‐α) as a model for mammary cell inflammation, we report that inhibition of HDACs1 and 2 (HDAC1/2) attenuated TNF‐α‐mediated inflammatory gene expression. Of note, we report that HDAC1/2‐mediated inflammatory gene expression was partly regulated by c‐Jun N‐terminal kinase (JNK) and extracellular signal‐regulated kinase (ERK) phosphorylation. Here, we report that HDAC1/2 inhibition attenuated JNK and ERK activation and thus inflammatory gene expression. These data suggest that HDACs1 and 2 regulate inflammatory gene expression via canonical (i.e., gene expression) and noncanonical (e.g., signaling dependent) mechanisms. Whereas, further studies using primary cell lines and animal models are needed. Our combined data suggest that HDAC1/2‐specific inhibitors may prove efficacious for the treatment of bovine mastitis.

Dietary protein reduction on microbial protein, amino acids digestibility, and body retention in beef cattle. I. Digestibility sites and ruminal synthesis estimated by purine bases and 15N as markers

The objectives of this study were to evaluate the effect of reducing dietary CP contents on 1) total and partial nutrient digestion and nitrogen balance and 2) on microbial crude protein (MCP) synthesis and true MCP digestibility in the small intestine obtained with 15N and purine bases (PB) in beef cattle. Eight bulls (4 Nellore and 4 Crossbred Angus × Nellore) cannulated in the rumen and ileum were distributed in duplicated 4 × 4 Latin squares. The diets consisted of increasing CP contents: 100, 120, or 140 g CP/kg DM offered ad libitum, and restricted intake (RI) diet with 120 g CP/kg DM. The experiment lasted four 17-d periods, with 10 d for adaptation to diets and another 7 for data collection. Omasal digesta flow was obtained using Co-EDTA and indigestible NDF (iNDF) as markers, and to estimate ileal digesta flow only iNDF was used. From days 11 to 17 of each experimental period, ruminal infusions of Co-EDTA (5.0 g/d) and 15N (7.03 g of ammonium sulfate enriched with 10% of 15N atoms) were performed. There was no effect of CP contents (linear effect, P = 0.55 and quadratic effect, P = 0.11) on ruminal OM digestibility. Intake of CP linearly increased (P < 0.01) with greater dietary CP. The NH3-N (P < 0.01) and urinary N excretion (P < 0.01) increased in response to dietary CP, whereas retained N increased linearly (P = 0.03). Liquid-associated bacteria (LAB) in the omasum had greater N content (P < 0.05) in relation to the particle-associated bacteria (PAB). There was no difference between LAB and PAB (P = 0.12) for 15N:14N ratio. The 15N:14N ratio was greater (P < 0.01) in RI animals in relation to those fed at voluntary intake. Microbial CP had a quadratic tendency (P = 0.09) in response to CP increase. Microbial efficiency (expressed in relation to apparent ruminally degradable OM and true ruminally degradable OM) had a quadratic tendency (P = 0.07 and P = 0.08, respectively) to CP increasing and was numerically greatest at 120 g CP/kg DM. The adjusted equations for estimating true intestinal digestibility of MCP (Y1) and total CP (Y2) were, respectively, as follows: Y1 =--16.724(SEM = 40.06) + 0.86X(SEM = 0.05) and Y2 = -43.81(SEM = 49.19) + 0.75X(SEM = 0.05). It was concluded that diets with 120 g/kg of CP optimize the microbial synthesis and efficiency and ruminal ash and protein NDF digestibility, resulting in a better use of N compounds in the rumen. The PB technique can be used as an alternative to the 15N to estimate microbial synthesis.

Nutritional evaluation and ruminal fermentation patterns of kochia compared with alfalfa and orchardgrass hays and ephedra and cheatgrass compared with orchardgrass hay as alternative arid-land forages for beef cattle in two dual-flow continuous culture system experiments1

The objective was to evaluate the ruminal fermentation patterns of forage kochia (FK) compared with alfalfa hay (AH) and orchardgrass hay (OH) (Exp. 1), and ephedra (EPH) and immature cheatgrass (CG) compared with OH (Exp. 2), using a dual-flow continuous culture system. Two in vitro experiments were conducted, and in each experiment, treatments were randomly assigned to six dual-flow fermenters (1,223 ± 21 mL) in a replicated 3 × 3 Latin square design, with three consecutive periods of 10 d each, consisting of 7 d for diet adaptation and 3 d for sample collection. Each fermenter was fed a total of 72 g/d (DM basis) and treatments were as follows: Exp. 1: 1) 100% OH, 2) 100% AH, and 3) 100% dried FK. Exp. 2: 1) 100% OH, 2) 100% dried CG, and 3) 100% dried EPH. On day 8, 9, and 10, samples of solid and liquid effluent from each fermenter were taken for digestibility analysis, and subsamples were collected for NH3-N, VFA, and bacterial N determinations. Data were analyzed using the MIXED procedure of SAS. In Exp. 1, treatments did not affect DM, OM, and NDF digestibilities, total VFA and molar proportions of acetate, propionate, butyrate, and branched-chain VFA. True CP digestibility, ruminal NH3-N concentration, and total N, NH3-N, NAN, and dietary N flows (g/d) were greater (P < 0.05) for FK compared with the other forages. However, treatments did not affect bacterial efficiency. In Exp. 2, DM, OM, and CP digestibilities were greater (P = 0.01) for EPH, and NDF digestibility was greater (P < 0.05) for EPH and CG compared with OH. Ephedra had the highest (P < 0.05) pH and acetate:propionate ratio and the lowest (P < 0.05) total VFA concentration. Total VFA, ruminal NH3-N concentration, and NH3-N flow (g/d) were highest (P < 0.05) for CG. Total N flow and bacterial efficiency were highest (P < 0.05) for OH and CG, while the flows (g/d) of NAN, bacterial N, and dietary N were greater (P < 0.05) for OH compared with the other forages. Results indicate that when compared with AH and OH (Exp. 1), FK has similar ruminal fermentation patterns and may be an adequate alternative for beef cattle producers. Furthermore, when compared with OH (Exp. 2), immature CG may also be an adequate forage alternative. This is especially important for areas in which conventional forages may not grow well such as the U.S. arid-land. However, EPH should not be used as the sole forage due to its poor ruminal fermentation as evidenced by the lowest total VFA concentration and propionate molar proportion.