Adaptation of in vitro methodologies to estimate the intestinal digestion of lipids in ruminants

The objective of this study was to adapt existing in vitro methodologies to determine the extent of intestinal digestion of corn oil (CO), canola oil (CA), and beef tallow (BT) via manipulation of incubation length and concentrations of lipase, bile, and calcium within a buffer solution. Unless otherwise stated, 0.5 g of each lipid source were incubated separately and in triplicate, with triplicate batch culture runs for each treatment in 40 mL of 0.5 M KH2PO4 (pH = 7.6) for 24 h with pancreatin (8 g/L), bovine bile (2.5 g/L), and CaCl2 (10 mM). Individually, concentrations of pancreatin, bile, and CaCl2, as well as incubation length were tested. To examine the use of this assay to estimate in vitro total tract digestion, a KH2PO4 solution with concentrated amounts to reach the same final concentrations of pancreatin, bile, and Ca were used as the third step in a three-step total tract digestibility procedure. Free glycerol and free fatty acid (FFA) concentrations were measured using colorimetric assays as indicators of digestion. Data wereanalyzed as a completely randomized block design (block = run), using the Glimmix procedure of SAS. For each lipid source, free glycerol increased with increasing pancreatin; however, FFA was lowest at 0 g/L pancreatin but was similar at 6, 8, and 10 g/L. Both glycerol and FFA were greater for 2.5 and 5 g/L of bile than for 0 g/L for each lipid source. Calcium concentration did not affect glycerol or FFA for either CO or CA; however, glycerol and FFA for BT were greater when calcium was included at 5 and 10 mM than at 0 mM. For all fat sources, free glycerol and FFA increased after 1 h until 12 h, but did not increase from 12 to 24 h. When a concentrated mixture was used following fermentation and acidification steps, digestibility using FFA concentration increased as compared to just adding buffer; however, free glycerol concentration was indeterminable. Thus, free glycerol and FFA can be used as indicators of lipid digestion when a lipid source is incubated for at least 12 h in a buffer solution containing 8 g/L pancreatin, 2.5 g/L bile, and 5 mM Ca when only estimating in vitro intestinal digestion; however, when utilizing this assay in a three-step in vitro total tract digestibility procedure, only FFA can be used.UCR::Vicerrectoría de Docencia::Ciencias Agroalimentarias::Facultad de Ciencias Agroalimentarias::Escuela de Zootecni

Ruminal Lipid A Analysis by Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry

Lipopolysaccharides (LPS) are cell wall components from Gram-negative bacteria and are composed of three covalently linked regions: the O-antigen, the core oligosaccharide, and the lipid A moiety, which carries most of their endotoxic activity. The objective of this study was to isolate and compare the lipid A structures from ruminal LPS derived from total mixed ration (TMR)- and pasture-fed cows, by using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Ruminal bacteria were collected from two rumen-cannulated Holstein cows; one fed a TMR (60:40, forage–concentrate) and the other pasture fed. The representativeness of each sample was validated by comparing the rumen microbiome from the cows in our study to the core rumen microbiome from the previous literature. Lipopolysaccharides from each respective sample were extracted with a phenol–water extraction procedure and purified via ultracentrifugation. To isolate lipid A from the core and O-antigen, pure ruminal LPS samples were hydrolyzed with acetic acid. Lipid A derived from the TMR-fed cow potentially exhibited a tetra-acylated structure, whereas lipid A derived from the pasture-fed cow potentially exhibited a penta-acylated lipid A structure. Both samples were quantified using limulus amebocyte lysate (LAL) assay and exhibited low endotoxic activity, consistent with the MALDI-TOF MS observations. Results indicate that the lipid A acylation pattern differs between diets, and that ruminal bacteria express solely under-acylated lipid A structures contrary to hexa-acylated lipid A, typically expressed by bacteria such as E. coli

Effects of supplemental source of magnesium and inclusion of buffer on ruminal microbial fermentation in continuous culture

José Alberto Arce Cordero realizó esta publicación durante un programa de doctorado patrocinado por la Universidad de Costa RicaMagnesium oxide (MgO) is the most common supple mental source of Mg for dairy cows and a proven ruminal alkalizer when supplemented above NRC (2001) recommendations. However, overfeeding MgO may increase feeding costs, whereas the effects of alternative sources of Mg on ruminal fermentation are not well known. Moreover, it is still unclear if Mg supplementation influences the effects of bicarbonate-based buffers on ruminal fermentation. We aimed to evaluate the effect of Mg source on ruminal fermentation with diets formulated to a final concentration of 0.25% Mg, and to determine if the effect of sodium sesquicarbonate as a buffer varies with the source of Mg. We used 8 fermentors in a duplicated 4 × 4 Latin square design with a 2 × 2 factorial arrangement of treatments, by combining 2 factors: (1) Mg source: using either MgO or an alternative source consisting of a blend of CaMg(OH)4 and CaMg(CO3)2 (BLN) and (2) sodium sesquicarbonate buffer inclusion, at 0 or 0.6% of dry matter intake. Based on preliminary tests of reactivity, we hypoth esized that BLN plus buffer would allow for greater ruminal pH, acetate molar proportion, and NDF digestibility than diets with MgO or without buffer. Four 10-d periods were completed, where the last 3 d were used for pH measurements and collection of samples for volatile fatty acids (VFA), ammonia (NH3-N), Mg solubility, N metabolism, and nutrient digestibility. Effects of Mg source (source), sodium sesquicarbonate inclusion (buffer), and their interaction (source × buffer) were tested with the MIXED procedure of SAS (SAS Institute Inc.). We did not find an effect of Mg source on ruminal fermentation variables; however, concentration of soluble Mg in ruminal fluid was greater for MgO compared with BLN. On the other hand, buffer supplementation increased average ruminal pH, acetate molar proportion, and branched-chain VFA molar proportion; tended to increase NDF digestibility; and decreased both area under the curve and time below pH 6.0. An interaction of source × buffer was found for propionate, butyrate, and NH3-N, the first one decreasing and the 2 others increasing only when buffer was supplemented to the BLN diet. Our results indicate that supplementing Mg with either MgO or BLN promotes similar ruminal fermentation in diets with total concentration of 0.25% Mg. Further evaluations are needed to assess Mg availability and animal performance in dairy cows fed BLN.Universidad de Costa Rica/[]/UCR/Costa RicaUCR::Vicerrectoría de Docencia::Ciencias Agroalimentarias::Facultad de Ciencias Agroalimentarias::Escuela de Zootecni

Megasphaera elsdenii and Saccharomyces Cerevisiae as direct fed microbials during an in vitro acute ruminal acidosis challenge

This study aimed to evaluate the efects of Saccharomyces cerevisiae and Megasphaera elsdenii as direct fed microbials (DFM) in beef cattle fnishing diets to alleviate acute ruminal lactic acidosis in vitro. A dual-fow continuous culture system was used. Treatments were a Control, no DFM; YM1, S. cerevisiae and M. elsdenii strain 1; YM2, S. cerevisiae and M. elsdenii strain 2; and YMM, S. cerevisiae and half of the doses of M. elsdenii strain 1 and strain 2. Each DFM dose had a concentration of 1 × ­108 CFU/mL. Four experimental periods lasted 11 days each. For the non-acidotic days (day 1–8), diet contained 50:50 forage to concentrate ratio. For the challenge days (day 9–11), diet contained 10:90 forage to concentrate ratio. Acute ruminal acidosis was successfully established. No diferences in pH, d-, l-, or total lactate were observed among treatments. Propionic acid increased in treatments containing DFM. For N metabolism, the YMM treatment decreased protein degradation and microbial protein synthesis. No treatment efects were observed on NH3–N concentration; however, efciency of N utilization by ruminal bacteria was greater than 80% during the challenge period and NH3–N concentration was reduced to approximately 2 mg/dL as the challenge progressed.UCR::Vicerrectoría de Docencia::Ciencias Agroalimentarias::Facultad de Ciencias Agroalimentarias::Escuela de Zootecni

In vitro evaluation of microencapsulated organic acids and pure botanicals as a supplement in lactating dairy cows diet on in vitro ruminal fermentation

The utilization of microencapsulated organic acids and pure botanicals (mOAPB) is widely used in the monogastric livestock industry as an alternative to antibiotics; in addition, it can have gut immunomodulatory functions. More recently, an interest in applying those compounds in the ruminant industry has increased; thus, we evaluated the effects of mOAPB on ruminal fermentation kinetics and metabolite production in an in vitro dual-flow continuous-culture system. For this study, two ruminal cannulated lactating dairy Holstein cows were used as ruminal content donors, and the inoculum was incubated in eight fermenters arranged in a 4 × 4 Latin square design. The basal diet was formulated to meet the nutritional requirements of a 680-kg Holstein dairy cow producing 45 kg/d of milk and supplemented with increasing levels of mOAPB (0; 0.12; 0.24; or 0.36% of dry matter [DM]), which contained 55.6% hydrogenated and refined palm oil, 25% citric acid, 16.7% sorbic acid, 1.7% thymol, and 1% vanillin. Diet had 16.1 CP, 30.9 neutral detergent fiber (NDF), and 32.0 starch, % of DM basis, and fermenters were fed 106 g/d split into two feedings. After a 7 d adaptation, samples were collected for 3 d in each period. Samples of the ruminal content from the fermenters were collected at 0, 1, 2, 4, 6, and 8 h postmorning feeding for evaluation of the ruminal fermentation kinetics. For the evaluation of the daily production of total metabolites and for the evaluation of nutrient degradability, samples from the effluent containers were collected daily at days 8 to 10. The statistical analysis was conducted using MIXED procedure of SAS and treatment, time, and its interactions were considered as fixed effects and day, Latin square, and fermenter as random effects. To depict the treatment effects, orthogonal contrasts were used (linear and quadratic). The supplementation of mOAPB had no major effects on the ruminal fermentation, metabolite production, and degradability of nutrients. The lack of statistical differences between control and supplemented fermenters indicates effective ruminal protection and minor ruminal effects of the active compounds. This could be attributed to the range of daily variation of pH, which ranged from 5.98 to 6.45. The pH can play a major role in the solubilization of lipid coat. It can be concluded that mOAPB did not affect the ruminal fermentation, metabolite production, and degradability of dietary nutrients using an in vitro rumen simulator.Universidad de Costa Rica/[739-C2-780]/UCR/Costa RicaUCR::Vicerrectoría de Docencia::Ciencias Agroalimentarias::Facultad de Ciencias Agroalimentarias::Escuela de Zootecni

Can dietary magnesium sources and buffer change the ruminal microbiota composition and fermentation of lactating dairy cows?

Magnesium oxide (MgO) is one of the most used Mg supplements in livestock. However, to avoid relying upon only one Mg source, it is important to have alternative Mg sources. Therefore, the objective of this study was to evaluate the effects of the interaction of two Mg sources with buffer use on the ruminal microbiota composition, ruminal fermentation, and nutrient digestibility in lactating dairy cows. Twenty lactating Holstein cows were blocked by parity and days in milk into five blocks with four cows each, in a 2 × 2 factorial design. Within blocks, cows were assigned to one of four treatments: 1) MgO; 2) MgO + Na sesquicarbonate (MgO+); 3) calcium–magnesium hydroxide (CaMgOH); 4) CaMgOH + Na sesquicarbonate (CaMgOH+). For 60 d, cows were individually fed a corn silage-based diet, and treatments were top-dressed. Ruminal fluid was collected via an orogastric tube, for analyses of the microbiota composition, volatile fatty acids (VFA), lactate, and ammonia nitrogen (NH3–N). The microbiota composition was analyzed using V4/16S rRNA gene sequencing, and taxonomy was assigned using the Silva database. Statistical analysis was carried out following the procedures of block design analysis, where block and cow were considered random variables. Effects of Mg source, buffer, and the interaction between Mg Source × Buffer were analyzed through orthogonal contrasts. There was no interaction effect of the two factors evaluated. There was a greater concentration of NH3–N, lactate, and butyrate in the ruminal fluid of cows fed with CaMg(OH)2, regardless of the buffer use. The increase in these fermentation intermediates/ end-products can be explained by an increase in abundance of micro-organisms of the genus Prevotella, Lactobacillus, and Butyrivibrio, which are micro-organisms mainly responsible for proteolysis, lactate-production, and butyrate-production in the rumen, respectively. Also, dietary buffer use did not affect the ruminal fermentation metabolites and pH; however, an improvement of the apparent total tract digestibility of dry matter (DM), organic matter (OM), neutral fiber detergent (NDF), and acid fiber detergent (ADF) were found for animals fed with dietary buffer. In summary, there was no interaction effect of buffer use and Mg source, whereas buffer improved total tract apparent digestibility of DM and OM through an increase in NDF and ADF digestibility and CaMg(OH)2 increased ruminal concentration of butyrate and abundance of butyrate-producing bacteria.UCR::Vicerrectoría de Docencia::Ciencias Agroalimentarias::Facultad de Ciencias Agroalimentarias::Escuela de Zootecni