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† Current address: Animal Production and Biotechnology group, Institute of Animal Health and Production, Universidad de Las Palmas de Gran Canaria, 35413 Arucas, Spain.
† Current address: Animal Production and Biotechnology group, Institute of Animal Health and Production, Universidad de Las Palmas de Gran Canaria, 35413 Arucas, Spain.
Affiliations
Department of Animal Science, Aarhus University, AU-Foulum, 8830 Tjele, Denmark
Department of Animal Science, Aarhus University, AU-Foulum, 8830 Tjele, DenmarkLinking Landscape, Environment, Agriculture and Food, Instituto Superior de Agronomia, Universidade de Lisboa, 13409-017 Lisbon, Portugal
† Current address: Animal Production and Biotechnology group, Institute of Animal Health and Production, Universidad de Las Palmas de Gran Canaria, 35413 Arucas, Spain.
The aim of this study was to investigate the effect of controlled daily alternations in dietary starch level on changes in rumen environment, blood, urine, and milk metabolites of dairy cows. Six multiparous mid-lactation Holstein cows were used in a replicated 3 × 3 Latin square design with 14-d periods and 3 alternating levels of dietary starch as treatments. Each 14-d period consisted of a 7-d baseline period and 7-d alternating period where diets alternated day to day. During the baseline period, all cows were fed a control diet containing 21% starch (dry matter basis). During the alternating period, the control diet was replaced with 1 of the 3 experimental diets on d 8, 10, 12, and 14. The 3 experimental diets contained 28% (low), 35% (medium), and 42% (high) starch (dry matter basis). At d 7 (baseline), 8 (ALT1), and 14 (ALT4) of each period, rumen fluid, blood, urine, and quarter milk (i.e., back right quarter) samples were collected at −0.5, 1, 2.5, 4, 5.5, and 7 h relative to morning feeding (0800 h). No differences were observed in dry matter intake, milk yield, and milk chemical composition. Rumen medial pH was lower in the high alternation level compared with the low or medium alternation levels at ALT1 but did not differ among starch alternation levels at ALT4. Similarly, the difference between rumen pH in medial and ventral contents was reduced at ALT1 with high alternation level but was not affected at ALT4. Total volatile fatty acid (VFA) concentrations were higher in the rumen medial fluid of the high alternation level at 7 h relative to morning feeding compared with those from the low and medium alternation levels. Similarly, total VFA concentrations constantly increased and were the highest in the ventral rumen fluid at 7 h relative to morning feeding, although no differences were detected among starch alternation levels. In both rumen medial and ventral fluids, the high alternation level showed higher propionate and lower acetate proportions compared with low and medium alternation levels. No differences in blood pH were detected among starch alternation levels. However, glucose concentrations tended to be higher in cows from the high alternation level. l-Lactate concentrations in blood were higher in ALT1 than in ALT4 but were not affected by the starch alternation level. In urine, no differences in pH or l-lactate concentrations were detected among alternation levels (i.e., low, medium, and high). Similarly, no differences in milk pH were detected among alternation levels. According to these results, it seems that the daily dietary starch alternation from 21% up to 42% (dry matter basis) is able to affect the ruminal fluid, especially during the first alternation. However, these changes in rumen fluid did not cause any effect on the variables measured in blood, urine, or milk. This study indicates that cows can cope with day-to-day alternations in type of rumen fermentable organic matter; however, longer-term effects on performance and health should be addressed in future studies.
). During lactation, dairy cows are commonly fed large amounts of rapidly fermentable carbohydrates such as starch, sugar, or pectins to sustain high milk yield. This affects the diet content of fermentable organic matter (FOM), which results in a shift in the rumen microbial population and increased production of VFA and lactate. Increased production of VFA and lactate increases the risk to exceed the absorption and buffering capacity of the rumen, which results in decreased rumen pH (
). In addition, composition of both concentrates and forages can change due to diverse factors such as plant genetics, growing conditions, harvesting, storing method, and manufacturing differences, among others. This, combined with imprecision in feedstuff weighing and time during ration mixing, might cause short-term changes in DM and nutrient content of the allocated feed (
In cows, a specific threshold for SARA has never been agreed upon by the scientific community. Some authors described that SARA is characterized by daily periods where ruminal pH is moderately reduced to 5.0 to 5.8 (
). In addition, SARA can negatively affect both animal health and performance by reducing feed intake, causing fluctuations in feed intake, reducing diet digestibility, decreasing milk yield and milk fat content, and increasing the risk for rumen epithelial ulcers, liver abscesses, and lameness (
). Due to the negative consequences of SARA in dairy farming, multiple studies have been performed to either enhance the knowledge about this condition or reduce its incidence in dairy cows (
The effects of starch and rapidly degradable dry matter from concentrate on ruminal digestion in dairy cows fed corn silage-based diets with fixed forage proportion.
). However, there is limited research on short-term changes (i.e., daily alternation) in diet composition on the rumen environment and production available in the literature, and it could be speculated that day-to-day alternations in type of FOM represent a risk factor for SARA.
Therefore, this study hypothesizes that daily alternations of the dietary starch level will induce fluctuating rumen pH and fermentation patterns in dairy cows. Based on this hypothesis, this study aimed to investigate the effect of controlled daily alternations in dietary content of barley and sugar beet pulp on changes in metabolite concentrations in rumen fluid, blood, urine, and milk.
MATERIALS AND METHODS
This experiment complied with the Danish Ministry of Justice Law No. 474 (May 15, 2014), concerning experiments with animals and care of experimental animals. The experimental protocol (15–12–02189) was described before the study was conducted.
Animals and Experimental Design
Six multiparous mid-lactation Holstein cows (BW 702 ± 61 kg; 150 ± 49 DIM at the beginning of the experiment) with permanent rumen cannulas and intercostal artery catheters were used in a replicated 3 × 3 Latin square design with 14-d periods and 3 levels of alternating dietary starch as treatments. Cows were housed in tiestalls bedded with mattresses and wood shavings. Cows were fed ad libitum once daily and had free access to water.
Each 14-d period consisted of a 7-d baseline period and 7-d alternating period where diets alternated day to day as illustrated in Figure 1. During the baseline period, all cows were fed a control diet containing 21% starch (DM basis; Table 1). During the alternating period, the control diet was replaced with 1 of the 3 experimental diets on d 8, 10, 12, and 14. The 3 experimental diets contained on a DM basis 28% starch (low), 35% starch (medium), or 42% starch (high). The increasing starch level was achieved by substituting dried sugar beet pulp with rolled barley (Table 1). The high Ca content of sugar beet pulp was balanced with limestone when substituted with barley. The following baseline period was also used to wash out the possible effects caused by the previous alternating period.
Figure 1Feeding sequences in each experimental treatment period showing the different starch alternation levels: low (from 21 to 28% of DM; black circles), medium (from 21 to 35% of DM; gray triangles), and high (from 21 to 42% of DM; white triangles). WIP = week in period.
Mineral and vitamin premix (VM 1; Vilofoss) containing (per kg): 165 g of Ca, 85 g of Mg, 100 g of Na, 40 g of S, 900 kIU of vitamin A, 190 kIU of vitamin D, 6,000 kIU of vitamin E, 4,000 mg of Mn, 1,500 mg of Cu, 25 mg of Co, 4,500 mg of Zn, 225 mg of I, and 50 mg of Se.
Calculated using the NorFor feed evaluation system at 20 kg of DMI/d (Volden, 2011).
MJ/kg DM
7.08
7.15
7.13
7.23
1 Low, from 21 to 28% of DM, n = 6; medium, from 21 to 35% of DM, n = 6; and high, from 21 to 42% of DM, n = 6.
2 Mineral and vitamin premix (VM 1; Vilofoss) containing (per kg): 165 g of Ca, 85 g of Mg, 100 g of Na, 40 g of S, 900 kIU of vitamin A, 190 kIU of vitamin D, 6,000 kIU of vitamin E, 4,000 mg of Mn, 1,500 mg of Cu, 25 mg of Co, 4,500 mg of Zn, 225 mg of I, and 50 mg of Se.
3 Calculated using the NorFor feed evaluation system at 20 kg of DMI/d (
Diet orts were removed and weighed daily at 0700 h. Subsequently, the daily portions of diets were weighed out for allocation at 0800 h and samples for chemical analysis were obtained. Cows were milked twice daily at 0630 and 1700 h. Milk yield was recorded at each milking using proportional continuous flow meters (Tru-Test WB/Pullout, Tru-Test Scandinavia A/S). Milk samples (50 mL) were collected from the continuous flow sample at the 2 following milkings after morning feeding on d 7, 8, and 14 (i.e., afternoon milking and morning milking next day). Water meters for recording of drinking water intake were read daily at morning milking. An experimental day was then defined as the time between 2 diet orts recordings (i.e., from 0700 h to 0700 h the next day).
At d 7, 8, and 14 of each period, rumen, blood, urine, and quarter milk (i.e., back right quarter) samples were collected at −0.5, 1, 2.5, 4, 5.5, and 7 h relative to morning feeding. Sampling at d 7 represented the baseline for all alternation levels (i.e., low, medium, and high) whereas sampling at d 8 and 14 represented the first and fourth daily starch alternations (i.e., ALT1 and ALT4, respectively). Medial rumen fluid was obtained from a ruminal mat sample collected 10 to 15 cm beneath the rumen cannula. The ruminal mat sample was squeezed using one layer of cheese cloth and the resulting medial rumen fluid was collected into a test tube (50 mL). Ventral rumen fluid was obtained by a 50-mL syringe attached to a suction strainer (#RT rumen fluid sampler tube, Bar Diamond Inc.) inserted into the ventral rumen through the cannula. The ventral rumen fluid sample was transferred to a test tube (50 mL). The pH in medial and ventral rumen fluids samples was measured immediately and subsequently an 8-mL subsample from both medial and ventral rumen fluid samples was stabilized by adding 2 mL of 25% metaphosphoric acid and then stored at −20°C until analysis. Arterial blood samples were collected through the intercostal catheter using disposable syringes and transferred to heparin Vacuettes (no. 455051, Greiner BioOne GmbH). Additional arterial samples were collected on heparinized 2-mL gas syringes for blood gas and oximetry measurements (PICO50, Radiometer A/S). Arterial blood samples were placed on ice immediately after collection. Then, blood samples were centrifuged at 3,000 × g for 20 min at 4°C and the resulting plasma was aliquoted and stored at −20°C until analysis. Urine samples (50 mL) were collected after stimulation by hand sweeping the supramammary region according to the methodology described by
Effect of time duration of ruminal urea infusions on ruminal ammonia concentrations and portal-drained visceral extraction of arterial urea-N in lactating Holstein cows.
. The resulting sample was placed on ice immediately after collection. Then, urine pH was measured, and a subsample (8 mL) was stored at −20°C until analysis. In addition, quarter milk samples (15 mL) were collected from the right back quarter by hand milking according to the methodology described by
Milk somatic cell count, lactate dehydrogenase activity, and immunoglobulin G concentration associated with mastitis caused by different pathogens: A field study.
. Then, milk pH was measured and a subsample (8 mL) was stored at −20°C until analysis.
Chemical Analysis
The DM content of the diets was determined by drying for 48 h at 60°C in a forced-air oven. Dried diet samples obtained at sampling days were analyzed for ash by combustion, for starch by enzymatic digestion, for CP by the Dumas principle, and for NDF using a Fibertec M6 System (Foss Analytical), all performed as described by
The pH in rumen fluid samples was measured using a combination electrode (PHC2002–8, Hach Lange ApS) and a pH meter (PHM240 pH/ION Meter, MeterLab, Radiometer analytical) calibrated at pH 4.005 and 7.000 (PHM 240, Hach Lange ApS). The stabilized rumen fluid samples were analyzed for concentrations of glucose and l-lactate using the immobilized glucose and l-lactate membrane oxidase electrode technique (YSI 2900D, YSI Inc.). Similarly, the concentrations of acetate (AC), propionate (PR), isobutyrate (iBU), butyrate (BU), isovalerate (iVAL), valerate (VALE), and caproate (CAP) were determined in the stabilized rumen fluid samples using GC following the methodology described by
Blood pH, blood gases, and oximetry variables were measured using an ABL700 Blood Gas Analyzer (Radiometer A/S). Hematocrit was determined immediately in arterial samples by centrifugation in capillary tubes at 13,000 × g for 6 min at room temperature (
Plasma samples were analyzed for glucose, l-lactate, and BHB using an autoanalyzer ADVIA 1800 Chemistry System (Siemens Medical Solution) according to the standard procedures described by the manufacturer (Siemens Diagnostics Clinical Methods for ADVIA 1800). d-Lactate was measured by an enzymatic-fluorometric method according to
Quantitation of blood and plasma amino acids using isotope dilution electron impact gas chromatography/mass spectrometry with U-C-13 amino acids as internal standards.
Rapid Commun. Mass Spectrom.1999; 13 (https://doi.org/10.1002/(SICI)1097-0231(19991115)13:21<2080::AID-RCM755>3.0.CO;2-O 10523763): 2080-2083
). A working AA standard solution was prepared from a commercial AA mixture (AAS18, Sigma-Aldrich Denmark A/S) with added Gln (l-Gln 99%, final concentration 400 µM, Acros), and urea (final concentration 7,500 µM, Merck). The internal standard was made from a U-13C/U-15N cell-free AA mixture (CNLM-6696–1, Cambridge Isotope Laboratories Inc.) with added [15N2] urea (no. 316830, Campro Scientific GmbH).
Urine pH was determined as described for rumen samples. Urine l-lactate and creatinine were determined according to standard procedures (Siemens Diagnostics Clinical Methods for ADVIA 1800). Analyses were performed using an autoanalyzer, ADVIA 1800 Chemistry System (Siemens Medical Solutions). d-Lactate was measured by an enzymatic-fluorometric method according to
Milk samples were analyzed for fat, protein, lactose monohydrate, and SCC by infrared spectrometry using a MilkoScan 4000 (Eurofins Steins A/S). Milk pH was measured as described for rumen samples. In the quarter milk samples, glucose, glucose 6P, isocitrate, BHB, glutamate, and malate were determined using an enzymatic-fluorometric method (excitation 544 nm, emission 590 nm) in a fluorometer (FLUOstar/Galaxy, BMG). Further details regarding the procedures for determining these variables can be found in previous publications from our group, specifically for glucose and glucose 6P (
), except that the mediating enzyme, reducing NAD+, was l-malate dehydrogenase (EC 1.1.1.37). Creatinine was determined according to standard procedures (ADVIA 1800, Siemens Diagnostics) after precipitation of protein and fat with ethanol, as indicated by the manufacturer.
Calculations and Statistical Analyses
Energy-corrected milk (3.140 MJ/kg) was calculated as described by
A Nordic proposal for an energy corrected milk (ECM) formula.
in: Performance Recording of Animals-State of the Art 1990. EAAP Publication 50. Centre for Agricultural Publishing and Documentation (PUDOC),
Wageningen, the Netherlands1991: 156-157
using the following equation: ECM yield (kg) = milk yield (kg) × [(38.3 × fat g/kg + 24.2 × protein kg/d + 15.7 × lactose g/kg + 20.7)/3.140].
All statistical analyses were performed using SAS software (version 9.3, SAS Institute Inc.) based on a linear mixed model with repeated measures (MIXED procedure). The model used to evaluate DMI during the entire experimental period included starch alternation level (from 21 to 28% vs. from 21 to 35% vs. from 21 to 42%), week in period (1 vs. 2), day within week in period (from d 1 to 7), and all possible interactions as dependent variables. Square and the interaction between cow and period were included as random effects. Day within cow, period, and week in period was set as the repeated subject. The model used to evaluate the rest of the variables measured in this study included starch alternation level (from 21 to 28% vs. from 21 to 35% vs. from 21 to 42%), day of alternation (ALT1 vs. ALT4), sampling time relative to morning feeding (from −0.5 to 7 h), and all possible interactions as dependent variables. Square and the interaction between cow and period were included as random effects. Sampling time within cow, period, and day of alternation was set as the repeated subject. Model assumptions were verified based on residual plots and tests for normality. Variables that were not normal distributed were log10 transformed to obtain normal distribution. A Tukey-Kramer test was used to evaluate differences among alternation levels. Values were considered significant when P ≤ 0.05. Results are presented as least squares means ± standard error of the mean.
RESULTS
DMI, Drinking Water Intake, and Milk Chemical Composition
Within each period (i.e., from d 1 to 14), daily DMI (Figure 2) did not differ among starch alternation level, week within period, or day within period week (P ≥ 0.31), although daily DMI numerically varied among starch alternation levels in the second week of the period compared with the first week.
Figure 2Daily DMI (PST×ALT > 0.05, where ST = starch and ALT = alternation) for low (from 21 to 28% of DM; black circles), medium (from 21 to 35% of DM; gray triangles), and high (from 21 to 42% of DM; white triangles) alternation levels; data are shown as LSM ± SE. WIP = week in period.
In addition, DMI and drinking water intake (Table 2) did not differ between alternation days (i.e., d 8 and 14) or among starch alternation levels (P ≥ 0.15). Similarly, milk yield and milk content of fat, protein, and lactose were not affected by the starch alternation level or day of alternation (P ≥ 0.12).
Table 2Feed intake, milk yield and milk composition for daily alternation (ALT) of starch (ST) at low (low, from 21 to 28% of DM; n = 6), medium (medium, from 21 to 35% of DM; n = 6), and high level (high, from 21 to 42% of DM; n = 6) at d 7 (baseline), 8 (ALT1), and 14 (ALT4) in each period
Rumen pH measured in the medial and ventral fluid as well as the difference between both are presented in Table 3 and Figure 3. A 3-way interaction between starch alternation level, day of alternation, and time was observed for medial rumen pH (PST×ALT×T = 0.04), with the lowest pH obtained in the high alternation level in ALT1 at 7 h relative to morning feeding (5.14 ± 0.13). Rumen ventral pH did not differ among starch alternation levels (P = 0.56) or day of alternation (P = 0.11) but decreased postprandial from 6.38 ± 0.07 to 5.90 ± 0.07 at 7 h relative to morning feeding (P < 0.01). A 2-way interaction between day of starch alternation and time after morning feeding was detected for the medial-ventral pH difference (PALT×T < 0.01), with the smallest difference observed in the ALT1 at 2.5 h relative to morning feeding (−0.46 ± 0.07), whereas the largest difference was observed in the ALT4 at 2.5 h relative to morning feeding (−0.71 ± 0.07).
Table 3Rumen pH in medial and ventral fluid as well as medial-ventral rumen pH difference for daily alternation (ALT) of starch (ST) at low (low, from 21 to 28% of DM; n = 6), medium (medium, from 21 to 35% of DM; n = 6), and high level (high, from 21 to 42% of DM; n = 6) measured postprandial (T, time) at d 7 (baseline), 8 (ALT1), and 14 (ALT4) in each period
Figure 3Ventral rumen pH and medial-ventral rumen pH difference for low (from 21 to 28% of DM; black circles), medium (from 21 to 35% of DM; gray triangles), and high (from 21 to 42% of DM; white triangles) alternation levels during daily starch alternation 1 (A; ALT1) and 4 (B; ALT4). The dashed red lines represent the baseline measured on d 7. Data are shown as LSM ± SE of samples collected at 1, 2.5, 4, 5.5, and 7 h after morning feeding.
Glucose, l-lactate, and VFA concentrations in medial rumen fluid are shown in Table 4. Glucose concentrations in medial rumen fluid increased postprandial (P < 0.01) from 0.08 ± 0.03 to 0.11 ± 0.03 mM at 7 h relative to morning feeding. l-Lactate concentrations in medial rumen fluid increased postprandial (P < 0.01) from 0.14 ± 0.16 to 0.64 ± 0.14 mM at 1.5 h, decreasing to 0.08 ± 0.14 mM at 7 h relative to morning feeding. A 2-way interaction between starch alternation level and time after morning feeding was observed for the total VFA concentration in medial rumen fluid (PST×T = 0.04), with the highest values obtained in cows from the high alternation level at 7 h relative to morning feeding (195 ± 8.0 mM). All the VFA proportions measured in the medial rumen fluid were affected by time after morning feeding (P < 0.01). Thus, proportions of AC and iBU constantly decreased postprandial, whereas proportions of PR, BU, iVAL, VALE, and CAP constantly increased postprandial. In addition, a 2-way interaction between starch alternation level and time after morning feeding interaction was observed for AC (PST×T = 0.01) and PR percentages (PST×T < 0.04). As shown in Figure 4A and 4B, cows from the high alternation level showed lower AC and higher PR proportions at both ALT1 and ALT4.
Table 4Medial rumen fluid variables for daily alternation (ALT) of starch (ST) at low (low, from 21 to 28% of DM; n = 6), medium (medium, from 21 to 35% of DM; n = 6), and high level (high, from 21 to 42% of DM; n = 6) measured postprandial (T, time) at d 7 (baseline), 8 (ALT1), and 14 (ALT4) in each period
Figure 4Butyrate (BU), propionate (PR), and acetate (AC) (mol/100 mol) in medial and ventral rumen for low (from 21 to 28% of DM; black circles), medium (from 21 to 35% of DM; gray triangles), and high (from 21 to 42% of DM; white triangles) alternation levels during daily starch alternation 1 (A and C; ALT1) and 4 (B and D; ALT4). The dashed red lines represent the baseline measured on d 7. Data are shown as LSM of samples collected at 1, 2.5, 4, 5.5, and 7 h after morning feeding ± SE. PST×T ≤ 0.05, where ST = starch and T = time.
Glucose, l-lactate, and VFA concentrations in ventral rumen fluid are shown in Table 5. Glucose concentrations in ventral rumen fluid were not affected by either the starch alternation level, day of starch alternation, or the time after morning feeding (P ≥ 0.20). l-Lactate concentrations in the ventral rumen increased postprandial (P < 0.01) from 0.09 ± 0.32 to 2.24 ± 0.32 mM at 1.5 h, decreasing to 0.37 ± 0.32 mM at 7 h relative to morning feeding. Similarly, total VFA concentration in ventral rumen fluid increased postprandial (P < 0.01) from 112 ± 3.6 to 135 ± 3.6 mM at 7 h relative to morning feeding. All the VFA proportions measured in the ventral rumen fluid were affected by time after morning feeding (P < 0.01). Thus, proportions of AC and iBU constantly decreased postprandial whereas proportions of PR, BU, iVAL, VALE, and CAP constantly increased postprandial. In addition, a 2-way interaction between starch alternation level and time after morning feeding was observed for the proportion of AC (PST×T = 0.03) and PR (PST×T = 0.04). As shown in Figure 4C and 4D, cows from the high alternation level showed lower AC and higher PR proportions at both ALT1 and ALT4.
Table 5Ventral rumen fluid variables for daily alternation (ALT) of starch (ST) at low (low, from 21 to 28% of DM; n = 6), medium (medium, from 21 to 35% of DM; n = 6), and high level (high, from 21 to 42% of DM; n = 6) measured postprandial (T, time) at d 7 (baseline), 8 (ALT1), and 14 (ALT4) in each period
Variables measured in arterial blood are shown in Table 6. Regarding the variables measured in whole blood, pH and the partial pressure of oxygen (pO2) were not affected by either starch alternation level (P = 0.71) or time after morning feeding (P = 0.13) and a trend for day of starch alternation was observed (P = 0.09). On the other hand, hematocrit percentage and the partial pressure of carbon dioxide (pCO2) were affected by the time after morning feeding (P ≤ 0.03). Hematocrit percentage decreased postprandial from 30.0 ± 0.51% to 28.8 ± 0.51% at 4 h, increasing to 29.0 ± 0.51 at 7 h relative to morning feeding. The pCO2 increased postprandial from 105 ± 7.33 to 130 ± 7.44 mm Hg at 1 h, decreasing to 119 ± 7.42 mm Hg at 2.5 h relative to morning feeding when pCO2 remained constant.
Table 6Blood variables for daily alternation (ALT) of starch (ST) at low (low, from 21 to 28% of DM; n = 6), medium (medium, from 21 to 35% of DM; n = 6), and high level (high, from 21 to 42% of DM; n = 6) measured postprandial (T, time) at d 7 (baseline), 8 (ALT1), and 14 (ALT4) in each period
Regarding the variables measured in plasma, l-lactate was affected by the daily starch alternation (P = 0.05), decreasing from the ALT1 (0.45 ± 0.03 mM) to the ALT4 (0.40 ± 0.03 mM). Plasma d-lactate concentrations were not affected by either starch alternation level, day of starch alternation, or time after morning feeding (P ≥ 0.12). Plasma glucose concentration tended (P = 0.09) to be higher in cows fed with a higher starch alternation level (4.02 ± 0.08 mM). Both glucose and BHB concentrations were affected by the time after morning feeding (P ≤ 0.01). Glucose concentrations decreased postprandial from 4.01 ± 0.07 to 3.85 ± 0.07 mM at 2.5 h, increasing to 4.03 ± 0.06 mM at 7 h relative to morning feeding. The concentration of BHB increased postprandial from 0.71 ± 0.03 to 0.91 ± 0.03 mM at 2.5 h, decreasing to 0.86 ± 0.03 mM at 7 h relative to morning feeding.
Regarding plasma AA concentrations, Asp and Glu were affected by the daily starch alternation (P ≤ 0.04), decreasing from ALT1 (7.10 ± 1.17 and 49.5 ± 5.92 µM, respectively) to ALT4 (6.44 ± 1.17 and 47.0 ± 5.92 µM, respectively). In addition, concentrations of Ala, Cys, Gly, Pro, Lys, Thr, Trp, and urea were affected by the time after morning feeding (P ≤ 0.05). Concentrations of Ala decreased postprandial from 208 ± 5.43 to 181 ± 5.41 µM at 7 h relative to morning feeding. Similarly, Cys concentrations decreased postprandial from 111 ± 2.83 µM to 110 ± 2.83 µM at 7 h relative to parturition. Concentrations of Gly decreased postprandial from 267 ± 10.3 to 240 ± 9.89 µM at 7 h relative to morning feeding. Concentrations of Pro decreased postprandial from 102 ± 2.11 to 87.7 ± 2.14 µM at 7 h relative to morning feeding. Concentrations of Lys decreased postprandial from 96.8 ± 8.10 to 85.6 ± 8.10 µM at 7 h relative to morning feeding. Concentrations of Thr decreased postprandial from 109 ± 6.17 to 98.4 ± 6.17 µM at 7 h relative to morning feeding. Concentrations of Trp decreased postprandial from 43.8 ± 2.15 to 41.1 ± 2.13 µM at 2.5 h, increasing to 44.1 ± 2.14 µM at 7 h relative to morning feeding. Urea concentrations increased postprandial from 3.48 ± 0.16 to 3.65 ± 0.16 mM at 1 h, decreasing to 3.09 ± 0.15 mM at 7 h relative to morning feeding. In addition, a 2-way interaction between starch alternation level and time after morning feeding was detected for Met and Thr concentrations (PST×T ≤ 0.03). Concentrations of Met remained constant in the low alternation level; however, they decreased postprandial in both medium and high alternation levels from 26.8 ± 2.31 to 20.5 ± 2.32 and from 26.8 ± 2.31 to 22.3 ± 2.31 at 7 h relative to morning feeding, respectively. Similarly, Thr concentrations remained constant in the low alternation level; however, they decreased postprandial in both medium and high alternation levels from 115 ± 8.10 µM to 99.5 ± 8.20, and from 106 ± 8.10 µM to 89.8 ± 8.10 µM at 7 h relative to morning feeding, respectively.
Urine Variables
Variables measured in urine are shown in Table 7. Urine pH was not affected by either starch alternation level or day of alternation (P ≥ 0.68) but decreased postprandial (P ≤ 0.01) from 8.17 ± 0.05 to 8.01 ± 0.05 at 4 h, remaining constant until 7 h relative to morning feeding (8.06 ± 0.05). Neither l-lactate, d-lactate, nor creatinine concentration was affected by starch alternation level, day of starch alternation, or time after morning feeding (P ≥ 0.21).
Table 7Urine variables for daily alternation (ALT) of starch (ST) at low (low, from 21 to 28% of DM; n = 6), medium (medium, from 21 to 35% of DM; n = 6), and high level (high, from 21 to 42% of DM; n = 6) measured postprandial (T, time) at d 7 (baseline), 8 (ALT1), and 14 (ALT4) in each period
Variables measured in milk are shown in Table 8. Milk pH as well as malate, creatinine, and isocitrate concentrations were not affected by either starch alternation level, day of starch alternation, or time after morning feeding (P ≥ 0.38). Glucose, BHB, glucose-6P, and glutamate concentrations were affected by the time after morning feeding (P ≤ 0.02). Glucose and glucose-6P concentrations in milk decreased postprandial from 308 ± 13.0 and 49.8 ± 8.92 µM to 132 ± 12.9 and 46.8 ± 8.94 µM at 4 h relative to morning feeding, respectively. These concentrations increased afterward to 171 ± 13.0 and 50.9 ± 8.93 µM at 7 h relative to morning feeding. Similarly, glutamate concentrations in milk decreased postprandial from 137 ± 9.18 µM to 33.4 ± 9.18 µM at 4 h relative to morning feeding, and subsequently increased to 84.7 ± 9.19 µM at 7 h relative to morning feeding. β-Hydroxybutyrate concentrations increased postprandial from 64.9 ± 4.82 to 98.1 ± 4.82 µM at 4 h and remained constant hereafter until 7 h relative to morning feeding (94.3 ± 4.82 µM).
Table 8Milk variables for daily alternation (ALT) of starch (ST) at low (low, from 21 to 28% of DM; n = 6), medium (medium, from 21 to 35% of DM; n = 6), and high level (high, from 21 to 42% of DM; n = 6) measured postprandial (T, time) at d 7 (baseline), 8 (ALT1), and 14 (ALT4) in each period
The effect of repeated and controlled daily alternations in type of dietary FOM on changes in rumen fluid, blood, urine, and milk metabolites of dairy cows were investigated to determine whether day-to-day variations in starch intake are a potential risk factor for SARA. Overall, the results showed that a high level of starch alternation induced ventral rumen pH that approached a moderately reduced level (5.0–5.8;
) only at the first day of alternation, but after 4 alternations, ventral rumen pH was above 6 during the first 7 h relative to morning feeding. In addition, no signs of systemic acidosis were observed as interpreted from blood and urinary pH.
Due to the energy requirements for milk production, high-yielding dairy cows are often fed rations with high energy concentrations. One of the most common ways of increasing dietary energy content is based on the inclusion of rapidly fermentable carbohydrates such as starch, which can promote an increase of the amylolytic and lactic acid bacteria populations, and in turn increases the production of VFA and lactate in the rumen (
Shifts in the rumen microbiota due to the type of carbohydrate and level of protein ingested by dairy cattle are associated with changes in rumen fermentation.
). In the current study, starch from barley grain was used to change the type of FOM while maintaining the level of dietary FOM relatively constant due to the high pectin content in sugar beet pulp (
Effect of dietary starch level and high rumen-undegradable protein on endocrine-metabolic status, milk yield, and milk composition in dairy cows during early and late lactation.
). However, few studies have focused on the effect of alternation in dietary starch on rumen fermentation and cow physiology and performance. In the present study, 4 day-to-day alternations were performed at 3 different dietary starch levels (i.e., from 21 to 28, from 21 to 35, and from 21 to 42%, DM basis). Both the starch alternation level and the daily starch alternation did not affect DMI. Although daily DMI was not affected, it is unknown whether the feeding pattern and the feed intake per hour were affected by either the starch alternation level or the daily starch alternation. Similarly, the starch alternation level and the daily starch alternation did not affect the concentrations of glucose and l-lactate in medial and ventral rumen fluids. However, increasing the dietary starch level caused a concomitant increase in the medial ruminal VFA concentrations, which in turn decreased pH in the medial rumen fluid during ALT1. The greater VFA concentration in medial rumen content at ALT1 was reflected in the concomitant reduced difference between acid load in medial and ventral rumen contents, indicating that the ventral pH became lower and closer to the critical levels. The reduced difference in pH levels was expected based on previous postprandial observations (
Effects of particle size and dry matter content of a total mixed ration on intraruminal equilibration and net portal flux of volatile fatty acids in lactating dairy cows.
Effects of forage neutral detergent fibre and time after feeding on medial and ventral rumen pH and volatile fatty acid concentration in heifers fed highly digestible grass/clover silages.
Effects of particle size and dry matter content of a total mixed ration on intraruminal equilibration and net portal flux of volatile fatty acids in lactating dairy cows.
, feeding low-fiber diets with high level of rapidly fermentable feed components results in lower ruminal stratification, increasing the intraruminal fluid flow and consequently creating a more homogeneous ruminal content. Surprisingly, the reduced difference in rumen pH between medial and ventral contents after feeding was not observed during the fourth daily alternation (ALT4). Based on the differences between ALT1 and ALT4, it seems that cows in the present study adapted to day-to-day starch alternations, even when high starch alternation levels were used (i.e., from 21 to 42% DM). As mentioned above, the similar FOM content among diets may have prevented SARA conditions from becoming established during repeated alternations in diet starch level. In addition, the increasing inclusion of limestone in the diets to compensate for the lower calcium content of barley compared with sugar beet pulp might have contributed to buffer rumen pH. However, the dissolving rate of limestone in ruminal conditions (i.e., temperature and pH) would be limited (
) and therefore it is unlikely that limestone inclusion affected ruminal pH.
Feeding diets rich in rapidly fermentable carbohydrates, such as starch, results in increased concentration of propionate in the rumen, whereas the relative concentration of butyrate and acetate decreases (
). Despite that the amount of FOM in the present study had a limited variation among experimental diets, the relative proportion of propionate progressively increased, and the relative proportions of acetate decreased with the increasing starch alternation level in both rumen fluids (i.e., medial and ventral) on both alternation days (i.e., ALT1 and ALT4). As described by
, the VFA proportions measured in the rumen are dependent on the rate at which monomer sugars are liberated from the substrate. Limited research is available comparing rumen fermentation of pectins with other easily fermentable carbohydrates, but
The effect of supplementing straw with barley or unmolassed sugar-beet pulp on microbial protein supply in sheep estimated from urinary purine derivative excretion.
observed increased VFA concentrations in rumen liquid when exchanging barley with sugar beet pulp in sheep. Altogether, limited differences are indicated in DM fermentation rate between sugar beet pulp and barley; however, more research is needed to describe the rumen fermentation pattern of pectins. Further, digesta passage rate could also have affected the amount of FOM, but passage rate did likely not differ substantially among treatments in the present study, as the major determinants of passage rate (i.e., DMI and forage type;
in: Sejrsen K. Hvelplund T. Nielsen M.O. Ruminant Physiology: Digestion, Metabolism and Impact of Nutrition on Gene Expression, Immunology and Stress. Wageningen Academic Publishers,
2006: 87-105
Although the increasing starch alternation level decreased rumen pH because of the concomitant increase in VFA concentrations in the rumen, pH values recorded in blood, urine, or milk were not affected by the starch alternation level in any of the 2 alternations tested (i.e., ALT1 and ALT4). Due to its importance in several physiological functions, blood pH is tightly regulated by the organism (
). d-Lactate is exclusively synthesized by microorganisms, especially lactobacilli, as a result of carbohydrate fermentation. Consequently, when high amounts of rapidly fermentable carbohydrates (e.g., starch) are fed to the cows, these microorganisms produce large amounts of d-lactate in the rumen (
), which contributes to decreased rumen pH. If large amounts of d-lactate are absorbed to the bloodstream through the rumen, blood pH decreases, causing a metabolic acidosis (
). As none of the animals used in the present experiment registered pH values in blood below 7.35, it can be considered that none of the tested starch alternation levels caused a metabolic acidosis, even when high starch alternations were used (i.e., from 21 to 42% DM). Indeed, the similar pO2, pCO2, and d-lactate concentrations registered among alternation levels confirms the previous statement.
Despite no significant differences in blood glucose were detected among alternation levels, this variable tended to increase with increasing dietary starch alternation level. As shown in this experiment, increasing starch alternation level caused a concomitant increase in the proportion of propionate in the rumen, which indicate an increased availability of propionate for liver gluconeogenesis. In addition, increasing starch level in the diet also increased the expected amount of digested rumen escape starch (
), which may have also contributed to increase circulating glucose concentration by direct glucose absorption from the small intestine.
Propionate absorbed to the portal venous blood is mostly taken up by the liver and contributes up to 60% of the hepatic glucose synthesized by the gluconeogenesis pathway and released into circulating blood (
in: Sejrsen K. Hvelplund T. Nielsen M.O. Ruminant Physiology: Digestion, Metabolism and Impact of Nutrition on Gene Expression, Immunology and Stress. Wageningen Academic Publishers,
2006: 225-248
). Gluconeogenesis could explain the tendency for higher blood glucose concentrations in cows from the high alternation level. In addition, similar concentrations of blood BHB observed among treatments can be explained by the lack of differences in the relative proportion of butyrate in both medial and ventral rumen fluid.
As described previously, cows suffering either clinical ruminal acidosis or SARA commonly decrease DMI, milk yield, and milk fat percentage (
, decreased availability of acetate and butyrate from rumen fermentation might cause a reduction in milk fat percentage. Also reduced rumen pH have been associated with reduced fatty acid biohydrogenation in the rumen, which increases the production of trans fatty acids (among others, trans-10 C-18:1 and trans-9,cis-11 CLA) and inhibits milk fat synthesis (
). In addition, increased rumen production of propionate results in increased blood glucose concentrations and therefore a concomitant reduction in mobilization of long-chain fatty acids from fat reserves (
). Despite the tendency for higher blood glucose concentrations observed in this study in those animals fed the high diet (i.e., from 21% to 42% starch level; DM basis), no differences in either milk yield or milk composition were detected among alternation levels. This indicates that none of the alternating diets tested in this study were able to induce a sufficient level of ruminal acidosis to cause changes in milk yield and composition. These results agree with
that observed no cumulative negative effects on DMI, milk yield, and milk components in dairy cows fed diets alternating in starch from 24 to 30% of DM for 5-d periods.
Effects of oscillating dietary crude protein concentrations on production, nutrient digestion, plasma metabolites, and body composition in lactating dairy cows.
observed how diets alternating from 12 to 16% CP (DM basis) reduced DMI without affecting milk yield. Overall, it seems that high-yielding dairy cows are relatively robust to deal with short-term variations in dietary components other than protein, especially when despite large changes in dietary starch level, FOM does not change to a great extent.
CONCLUSIONS
In this study, the daily alternation of dietary starch from 21% to 42% of DM increased the total concentration of VFA in the medial rumen fluid. In addition, the daily starch alternation from 21% to 42% of DM increased the proportion of propionate and decreased the proportion of acetate and butyrate in both medial and ventral rumen fluid. However, changes in the ruminal fluid did not affect the variables measured in blood, urine, and milk. Our results indicate that cows can cope with day-to-day alternations in type of rumen FOM; however, longer-term effects on performance and health should be addressed in future studies.
ACKNOWLEDGMENTS
The authors thank department staff Anne Krustrup (Aarhus University, Denmark), Birgit H. Loth (Aarhus University, Denmark), Ester Bjerregaard (Aarhus University, Denmark), and Torkild N. Jakobsen (Aarhus University, Denmark) for sampling and laboratory analysis, and Adam C. Storm (Novozymes, Denmark) for assisting during the intercostal catheter surgery. We also thank department barn staff for care and handling of experimental animals. The Danish Milk Levy Fund (Aarhus, Denmark) and Aarhus University (Aarhus, Denmark) provided funding for the study. Lorenzo E. Hernández-Castellano acknowledges financial support under the Ramón y Cajal programme (RYC2019-027064-I, Spain). The authors confirm that there were no conflicts of interest.
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