Effects of weaning age and pace on blood metabolites, cortisol concentration, and mRNA abundance of inflammation-related genes in gastrointestinal, adipose, and liver tissue of Holstein dairy calves.

The objective of this study was to evaluate the effects of weaning age and pace on blood metabolites, cortisol concentration, and mRNA abundance of inflammation-related genes in Holstein dairy calves. A total of 70 1-d-old calves (38.8 ± 4.4 kg BW ± SD), blocked by sex and birth BW, were randomly assigned to a 2 × 2 factorial arrangement of treatments. The first factor was weaning age, which was either early (6 wk) or late (8 wk). The second factor was weaning pace, which was either abrupt (4 steps down over 3 d; the initial milk replacer was 7.6 L, which was reduced by 1.9 L in each step-down) or gradual (7 steps down over 14 d; the initial milk replacer was 7.6 L, which was reduced by 1.09 L in each step-down), generating early-abrupt (EA), early-gradual (EG), late-abrupt (LA), and late-gradual (LG) treatments. All treatments had 10 female and 8 male calves, except EA that had 1 fewer male calf. Milk replacer (24% CP, 17% fat) was bottle fed, up to 1,200 g/d, twice daily (0600 h and 1800 h). The EA and EG treatment calves received 46.2 kg of milk replacer, and the LA and LG treatment calves received 63 kg of milk replacer. The study had 2 cohorts (2020, n = 40; 2021, n = 31), and each cohort included all treatments. Blood was collected from the jugular vein at 0900 h at 3 and 7 d of age, and a day before starting and a day after weaning completion. Male calves were humanely killed a day after weaning. Rumen, jejunum, large intestine, liver, omental adipose and perirenal adipose tissues were sampled to determine the mRNA abundance of inflammation-related genes. Weaning pace, age, pace × age, birth BW, and sex were included as fixed and cohort was included as random effects in the model. Blood metabolites and cortisol were analyzed as repeated measures, and sampling day, pace × sampling day, and age × sampling day were also included as additional fixed effects. Significance was noted at P ≤ 0.05 and tendencies when 0.05

Effect of pH and lipopolysaccharide on tight junction regulators and inflammatory markers in intestinal cells as an experimental model of weaning transition in dairy calves.

Acidic conditions combined with the presence of lipopolysaccharide (LPS) may increase the permeability of gastrointestinal epithelium. Feeding starch-rich starter to dairy calves is associated with ruminal acidosis and decreases the pH of other segments of the gastrointestinal tract, and that affects the tight junction regulator. The objective was to evaluate the effect of the combination of different pH (7.4 vs. 6.0) and LPS concentrations (0, 0.5, 10 ng/mL) in intestinal cells on tight junction regulators, inflammatory markers, and permeability. The human colon carcinoma Caco-2 cell line was used with the main treatment of pH and LPS in a 2 × 3 factorial arrangement. The pH was acidic (pH 6.0) or physiologic (pH 7.4), whereas LPS was 0, 0.5, or 10 ng/mL. After cells reached 70%-80% of confluence, the media were replaced with each respective treatment medium. Cells were treated for 3 h for mRNA abundance analysis, 3 and 6 h for protein abundance determination, and 3, 6, 12, and 24 h for permeability determination. Protein abundance of the myosin light-chain kinase (MYLK) and toll-like receptor 4 (TLR4) were measured by western blot. The mRNA abundance of IL-8, MYLK, peroxisome proliferator activated receptor gamma, and nuclear factor kappa B (Nfkb1) was determined by real-time, quantitative PCR. Paracellular permeability was determined with Lucifer yellow after 21 d of incubation. Cell culture was performed in biological triplicate; each biological replicate for real-time, quantitative PCR had 2 technical replicates, and for protein abundance and permeability assay had one technical replicate. The MIXED procedure of SAS (SAS Institute Inc.) was used with LPS, pH, and pH × LPS as fixed effects. Significance was declared at P ≤ 0.05 and tendencies when 0.05 < P ≤ 0.10. Increasing LPS doses did not affect the protein abundance of MYLK and TLR4, nor mRNA abundance of MYLK and PPRG. The LPS tended to increase mRNA abundance of IL-8 while pH × LPS interactively increased mRNA abundance of Nfkb1, where mRNA abundance of Nfkb1 was lower in cells exposed to pH 6.0 when combined with 0 and 10 ng/mL of LPS. Contrary to expectations, LPS did not affect the permeability of Caco-2 cells. The mRNA abundance of MYLK was greater at pH 6.0 versus pH 7.4. Also, protein abundance of TLR4 was lower at pH 6.0 than pH 7.4, and it decreased when exposure increased to 6 h. In addition, mRNA abundance of IL-8 was lower at pH 6.0 versus pH 7.4. Permeability was greater at pH 6.0 versus 7.4 after 6, 12, and 24 h of treatment. In summary, the effect of LPS and its interaction with pH showed less impact than expected on dependent variables measured, which might be attributed to the adopted clinically achievable LPS doses likely not being high enough to draw a strong response as observed in the literature. On the other hand, pH was far more relevant, modulating mRNA abundance of inflammatory markers, tight junction regulators, and permeability in in vitro colon cell models.

Effects of weaning strategies on health, hematology, and productivity in Holstein dairy calves.

Weaning strategies in dairy calves vary considerably, though the effect on animal health is unclear. This study examined the effects of calf weaning age (6 vs. 8 wk) and pace (abrupt vs. gradual) on performance, blood, and health parameters in dairy calves. The experiment consisted of a 2 × 2 factorial arrangement of treatments, where the factors included weaning age (early vs. late) and weaning pace (abrupt vs. gradual). Holstein calves (n = 72), blocked by sex and birth weight, were randomly assigned to one of 4 treatments (n = 18 per treatment): early-abrupt (EA), early-gradual (EG), late-abrupt (LA), and late-gradual (LG). Milk replacer (24% crude protein, 17% fat; up to 1,200 g/d) was fed twice daily; water, calf starter (18% crude protein), and chopped alfalfa hay were fed ad libitum. Daily intakes of milk replacer, calf starter, and forage were recorded from birth until end of weaning. Body weight, selected health measures, blood hematology, and fecal scores were obtained 1 d preweaning and 1 d postweaning. Calves were orally bolused with a rumen pH logger for the last 3 d of the weaning transition and rumen pH was measured continuously. Data were analyzed with age, pace, age × pace interaction, birthweight, and sex as fixed effects, and starting date as a random effect. Greater age at weaning increased respiration, whereas gradual-weaned calves had lower respiration rate. Heart rate was lower in gradual than in abrupt weaned groups. Fecal score had a marginal increase in late-weaned groups and significantly increased in gradually weaned groups. No difference was detected in body core temperature by age, pace, or interaction. During the weaning transition, average daily gain was lower in LA than EA and gradually weaned groups had an increased average daily gain. Change in grain intake, but not forage intake, was greater in gradually weaned groups. Mean rumen pH marginally increased from EG to LG and from LA to LG. No difference was detected among treatments in red or white blood cell counts, and hemoglobin. Procalcitonin was marginally highest in the LA group, while blood hematocrit increased in abruptly weaned groups. Overall, calf health is affected by both age and pace of weaning, though the health parameters affected by age and pace differ.

Production, physiological response, and calcium and magnesium balance of lactating Holstein cows fed different sources of supplemental magnesium with or without ruminal buffer.

The objective of this study was to evaluate the effects of dietary replacement of magnesium oxide (MgO) with calcium-magnesium hydroxide [CaMg(OH)2] and its interaction with ruminal buffer (sodium sesquicarbonate) supplementation on production, Ca and Mg balance, and overall physiological response of mid-lactation Holstein dairy cows. Sixty cows averaging 40.5 ± 7.0 kg of milk/d were used. Treatments were assigned following a 2 × 2 factorial arrangement: (1) MgO, (2) MgO + buffer, (3) CaMg(OH)2, or (4) CaMg(OH)2 + buffer. Diets were formulated to have 16.5% of crude protein, 1.82 Mcal/kg of net energy for lactation, 0.67% Ca, 0.39% P, and 0.25% Mg, all on a dry matter (DM) basis. Treatments were individually top dressed. Milk production, composition, and DM intake were evaluated. A subsample of 20 cows were randomly selected for the evaluation of Ca and Mg balance, blood gases, and electrolytes. Ruminal fluid was also collected for evaluation of pH and Ca and Mg solubility. Effects of Mg source, buffer, and the interaction Mg source × buffer were analyzed through orthogonal contrasts. An interaction of Mg source × buffer was found for DM intake and feed efficiency, in which cows fed CaMg(OH)2 had a similar feed efficiency regardless of ruminal buffer inclusion; however, when cows were fed MgO, the inclusion of buffer reduced feed efficiency. No effects on body weight and milk yield were observed. Buffer addition tended to increase the concentrations of fat, protein, and solids-not-fat, without affecting the yields of these milk components. Magnesium source and buffer did not affect ruminal fluid, blood, urine, or fecal pH; however, buffer supplementation increased urinary pH. Treatment with CaMg(OH)2 increased blood concentration of HCO3-, total CO2, and base excess compared with cows fed MgO. No differences were observed in the ruminal solubility of Ca and Mg or on milk or urinary Ca and Mg excretion. Greater plasma Mg concentration was observed for animals fed MgO compared with cows fed CaMg(OH)2; however, both sources were above the threshold recommended in the literature for dairy cows. Also, a reduction in fecal Mg excretion was observed in animals fed CaMg(OH)2. In summary, we provide evidence that CaMg(OH)2 could replace MgO without affecting performance, overall physiological response, or Ca and Mg balance of mid-lactating dairy Holstein cows.

Effects of exogenous amylolytic or fibrolytic enzymes inclusion on in vitro fermentation of lactating dairy cow diets in a dual-flow continuous-culture system.

The objective of this study was to determine the effects of including exogenous amylolytic or fibrolytic enzymes in a diet for high-producing dairy cows on in vitro ruminal fermentation. Eight dual-flow continuous-culture fermentors were used in a replicated 4 × 4 Latin square. The treatments were control (CON), a xylanase and glucanase mixture (T1), an α-amylase mixture (T2), or a xylanase, glucanase, and α-amylase mixture (T3). Treatments were included at a rate of 0.008% of diet dry matter (DM) for T1 and T2 and at 0.02% for T3. All treatments replaced the equivalent amount of soybean meal in the diet compared with CON. All diets were balanced to have the same nutrient composition [30.2% neutral detergent fiber (NDF), 16.1% crude protein (CP), and 30% starch; DM basis], and fermentors were fed 106 g/d divided into 2 feedings. At each feeding, T2 was pipetted into the respective fermentor and an equivalent amount of deionized water was added to each fermentor to eliminate potential variation. Experimental periods were 10 d (7 d for adaptation and 3 d for sample collection). Composite samples of daily effluent were collected and analyzed for volatile fatty acids (VFA), NH3-N, and lactate concentrations, degradability of DM, organic matter, NDF, CP, and starch, and flow and metabolism of N. Samples of fermentor contents were collected from each fermentor at 0, 1, 2, 4, 6, and 8 h after feeding to determine kinetics of pH, NH3-N, lactate, and VFA concentrations over time. All data were analyzed using PROC GLIMMIX of SAS (SAS Institute Inc.), and the repeated variable of time was included for kinetics measurements. Treatment did not affect mean pH, degradability, N flow and metabolism, or the concentrations of VFA, NH3-N, or lactate in the effluent samples. Treatment did not affect pH, acetate:propionate ratio, or the concentrations of lactate, NH3-N, total VFA, acetate, propionate, butyrate, isobutyrate, valerate, or caproate. However, the concentration of total VFA tended to change at each time point depending upon the treatment, and T2 tended to have a greater proportion of 2-methylbutyrate and isovalerate than CON, T1, or T3. As 2-methylbutyrate and isovalerate are branched-chain VFA that are synthesized from branched-chain amino acids, T2 may have an increased fermentation of branched-chain amino acids or decreased uptake by fibrolytic microorganisms. Although we did not observe changes in N metabolism due to the enzymes, there could be changes in microbial populations that utilize branched-chain VFA. Overall, the tested enzymes did not improve in vitro ruminal fermentation in the diet of high-producing dairy cows.

Effects of replacing magnesium oxide with calcium-magnesium carbonate with or without sodium bicarbonate on ruminal fermentation and nutrient flow in vitro.

The objective of this study was to evaluate the effects of replacing magnesium oxide (MgO) with calcium-magnesium carbonate [CaMg(CO3)2] on ruminal fermentation with or without the addition of sodium bicarbonate (NaHCO3). Eight fermentors of a dual-flow continuous-culture system were distributed in a replicated (2) 4 × 4 Latin square design in a 2 × 2 factorial arrangement of treatments (magnesium sources × NaHCO3). The treatments tested were 0.21% MgO [MgO; dry matter (DM) basis; 144.8 mEq of dietary cation-anion difference (DCAD)]; 0.21% MgO + 0.50% NaHCO3 (MgO+NaHCO3; DM basis; 205.6 mEq of DCAD); 1.00% CaMg(CO3)2 [CaMg(CO3)2; DM basis; 144.8 mEq of DCAD]; and 1.00% CaMg(CO3)2 + 0.50% NaHCO3 [CaMg(CO3)2+NaHCO3; DM basis; 205.6 mEq of DCAD]. Diets were formulated to have a total of 0.28% of Mg (DM basis). The experiment consisted of 40 d, which was divided into 4 periods of 10 d each, where 7 d were used for adaptation and 3 d for sampling to determine pH, volatile fatty acids (VFA), ammonia (NH3-N), lactate, mineral solubility, N metabolism, and nutrient digestibility. The effects of Mg source [MgO vs. CaMg(CO3)2], NaHCO3 (with vs. without), and the interaction were tested with the MIXED procedure of SAS version 9.4 (SAS Institute). There was no Mg source × NaHCO3 interaction in the pH variables and mineral solubility, and Mg sources evaluated did not affect the variables related to ruminal pH and solubility of Mg. On the other hand, the inclusion of NaHCO3 increased the pH daily average, independent of Mg source, which led to a reduced time that pH was below 5.8 and decreased area under the curve. Total VFA and lactate concentration were similar among treatments regardless of NaHCO3 and Mg source; however, the molar proportion of isobutyrate and NH3-N concentration were lower in diets with CaMg(CO3)2 compared with MgO. Moreover, NaHCO3 inclusion increased NH3-N, total daily NH3-N flow, isobutyrate concentration, and acid detergent fiber digestibility. Our results showed that CaMg(CO3)2 leads to a lower NH3-N concentration and isobutyrate proportion. Therefore, because most of the tested variables were not significantly different between MgO and CaMg(CO3)2 when combined or not with NaHCO3, CaMg(CO3)2 can be a viable alternative source to replace MgO in dairy cow diets without affecting mineral solubility, ruminal pH, nutrient digestibility, total VFA, and the main ruminal VFA. Although Mg sources are known to have an alkalizing effect, NaHCO3 inclusion in diets with Mg supplementation allowed an increase in ruminal pH, as well as an increase in isobutyrate and NH3-N flow.

Enzymatic effects of Pleurotus ostreatus spent substrate on whole-plant corn silage and performance of lactating goats.

Pleurotus ostreatus (oyster mushroom) synthesizes enzymes that degrade lignin, cellulose, and hemicellulose. The objectives of this study were to evaluate the effect of Pleurotus ostreatus spent substrate (POSS) on whole-plant corn silage (WPCS) chemical composition, antioxidant capacity, lignin monomers, and in vitro digestibility, as well as the performance of lactating goats fed corn silage treated with different levels of POSS. In experiment 1, 4 levels of lignocellulolytic enzymes were tested in a complete randomized design: 0, 10, 20, and 30 mg of lignocellulosic enzymes per kilogram of fresh matter, 4 replicates per treatment (vacuum-sealed bags). The bags were opened 60 d after ensiling. In experiment 2, corn silage treated with 3 enzyme levels (0, 10, or 30 mg/kg of fresh matter) was fed to lactating goats as part of the total mixed ration. Nine lactating Saanen goats (62.68 ± 7.62 kg BW; 44 ± 8 d in milk; 2.91 ± 0.81 kg of milk/day, mean ± SD) were assigned to three 3 × 3 Latin squares. Data were analyzed using the GLIMMIX procedure of SAS (version 9.4, SAS Institute Inc.), and means were compared by linear and quadratic orthogonal contrast. In experiment 1, neutral detergent fiber (NDF), acid detergent fiber (ADF), lignin, and cellulose quadratically decreased in the WPCS treated with POSS. At the nadir point, POSS decreased NDF by 14.1%, ADF by 19.5%, lignin by 9.07%, and cellulose by 22.1% compared with the untreated silage. Therefore, POSS led to a quadratic increase in in vitro dry matter digestibility of WPCS (+8.88% at the vertex) compared with the untreated silage. In experiment 2, POSS quadratically increased the in vivo total-tract ADF digestibility. Also, the concentration of polyphenols in the milk of goats linearly increased with the addition of POSS, and no differences were observed among treatments for milk yield and composition. In summary, adding 10 mg of lignocellulolytic enzymes from POSS per kilogram of fresh matter of whole-plant corn at ensiling had a more evident reduction in lignin and cellulose concentration, leading to greater in vitro digestibility, as well as greater in vivo ADF digestibility; however, milk yield was not different among treatments.

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

Magnesium oxide (MgO) is the most common supplemental 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 hypothesized 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.

Effects of bacterial cultures, enzymes, and yeast-based feed additive combinations on ruminal fermentation in a dual-flow continuous culture system.

Bacterial cultures, enzymes, and yeast-derived feed additives are often included in commercial dairy rations due to their effects on ruminal fermentation. However, the effects of these additives when fed together are not well understood. The objective of this study was to evaluate the changes in ruminal fermentation when a dairy ration is supplemented with combinations of bacterial probiotics, enzymes and yeast. Our hypotheses were that ruminal fermentation would be altered, indicated through changes in volatile fatty acid profile and nutrient digestibility, with the inclusion of (1) an additive, (2) yeast, and (3) increasing additive doses. Treatments were randomly assigned to 8 fermenters in a replicated 4 × 4 Latin square with four 10 d experimental periods, consisting of 7 d for diet adaptation and 3 d for sample collection. Basal diets contained 52:48 forage:concentrate and fermenters were fed 106 g of dry matter per day divided equally between two feeding times. Treatments were: control (CTRL, without additives); bacterial culture/enzyme blend (EB, 1.7 mg/d); bacterial culture/enzyme blend with a blend of live yeast and yeast culture (EBY, 49.76 mg/d); and a double dose of the EBY treatment (2×, 99.53 mg/d). The bacterial culture/enzyme blend contained five strains of probiotics (Lactobacillus animalis, Propionibacterium freudenreichii, Bacillus lichenformis, Bacillus subtilis, and Enterococcus faecium) and three enzymes (amylase, hemicellulase, and xylanase). On d 8-10, samples were collected for pH, redox, volatile fatty acids, lactate, ammonia N, and digestibility measurements. Statistical analysis was performed using the GLIMMIX procedure of SAS. Repeated measures were used for pH, redox, VFA, NH3-N, and lactate kinetics data. Orthogonal contrasts were used to test the effect of (1) additives, ADD (CTRL vs. EB, EBY, and 2X); (2) yeast, YEAST (EB vs. EBY, and 2X); and (3) dose, DOSE (EBY vs. 2X). No effects (P > 0.05) were observed for pH, redox, NH3-N, acetate, isobutyrate, valerate, total VFA, acetate:propionate, nutrient digestibility or N utilization. Within the 24 h pool, the molar proportion of butyrate increased (P = 0.03) with the inclusion of additives when compared to the control while the molar proportion of propionate tended to decrease (P = 0.07). In conclusion, the inclusion of bacterial cultures, enzymes and yeast in the diet increased butyrate concentration; but did not result in major changes in ruminal fermentation.