Acid-base balance in dairy cattle

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MATERIALS AND METHODS

Animals

Eight Jersey and seventeen Holstein cows were selected from the Virginia Tech Dairy Center and randomly divided into two diet treatment groups according to lactation, production, and days in milk. At the beginning of the experiment, Jerseys averaged a parity of 2.5, 26.9 kg/d milk, 91 days in milk (DIM), and 434 kg bodyweight. Holsteins averaged a parity of 2, 40.4 kg/d milk, 91.7 DIM, and 621 kg bodyweight. Cows were housed in free stalls and separated by breed. Cows were fed individually 100% of their daily ration at 0900h. Diets were fed for ad libitum intake, allowing for at least 10% refusal. Cows had access to water at all times except during milking. Cows were out of the free stall barns approximately 1 h for milking at 0145 h and 1245 h.

Diets

Four experimental dietary regimes were offered to cows during eight 10-d periods allowing for each regime to be replicated once. The first two dietary regimes were total mixed rations only, one containing no buffering agent (diet 1) and the other containing 1.2% sodium bicarbonate on a dry matter basis (diet 2). The remaining two dietary regimes were addition of free-choice options to the two total mixed rations. Sodium bentonite (Volclay, Arlington Heights, IL) and sodium bicarbonate (Church & Dwight; Division of Arm & Hammer Co.) were offered side by side in a covered feeder to breed groups during specific periods (Figure 1). Total mixed rations were formulated to contain 17.0% acid detergent fiber and 17.0% crude protein. They also contained 30% soluble protein, 72% total digestible nutrients (TDN), 34% starch, and 1.61 mcal/d NEL.
The ration was approximately 60% forage. Corn silage, alfalfa silage, high moisture corn, and 48% soybean meal were the main ingredients. Both diets contained approximately 50% dry matter. Particle size was analyzed using a Penn State Particle Size Separator with the top, middle, and bottom sieves containing 2.3, 33.0, and 64.7 % of the TMR, respectively. Percentages of dry matter and chemical analyses are in Table 1.

Experimental design

The study was conducted from September 19 through December 8, 2001. A total of eight 10-d periods were conducted with 8-d diet adjustment and 2-d sample collection. Every 2 periods (20 days) cows were switched to the alternate TMR (diet 1 or 2). During periods 2, 3, 5, and 8, free-choice options were offered to each breed group (Figure 2) and average group intake of sodium bentonite and sodium bicarbonate was recorded every 48 h during the period. Because only group intake was determined, it was not possible to establish individual cow intake of sodium bentonite or sodium bicarbonate. Diets were maintained at similar ADF and crude protein percentages throughout the trial.

Measurements and sampling

Samples of corn and alfalfa silages were sampled weekly and submitted to the Virginia Tech Forage Testing Laboratory for determination of dry matter, crude protein, and ADF to adjust the diets. Feed refusals were recorded daily for each cow and the amounts of ration adjusted accordingly to allow for optimum intake and to record daily intake. Daily intakes were used to calculate period averages. Milk production was measured daily throughout the 80 d trial (0130h, 1300h) to establish a period average. Milk samples were obtained from one morning and two consecutive afternoon milkings for component analysis. Milk was analyzed by the Virginia Dairy Herd Improvement Association for fat and protein percentages (MilkoScan 4000 series, Foss North America, Eden Prairie, MN) and somatic cell count (FM 5000, Foss North America, Eden Prairie, MN). MUN was analyzed (ChemSpec 150, Bently Instruments, Inc., Chaska, MN). Cows were weighed for three consecutive days: two days before the trial started, at the beginning of period 5, and at the end of the trial.
On d 10 of each period, samples of blood, urine, and manure were obtained between 1430 h and 1730 h. Blood was collected in evacuated heparinized centrifuge tubes via tail or jugular venipuncture and analyzed after collection. Hema tocrit samples (subsample drawn from the original blood sample into capillary tubes) were centrifuged at 13,700 x g (Autocrit Ultra3, Clay Adams) for 5 min and analyzed for packed cell volume (PCV).
Remaining blood was centrifuged at 3,600 x g for 10 min at 4oC to separate plasma. Plasma was collected and analyzed for protein levels by refractometer. Remaining blood plasma was stored at –20oC for later analysis. Urine was obtained by manual stimulation, refrigerated for 24 h, and warmed to room temperature prior to testing for pH by probe and specific gravity by refractometer. Fecal samples were obtained by rectal evacuation. Fecal pH (VXR digital model 2000, Orion Research, Inc.) was measured shortly after collection by inserting the pH probe directly into a sample of feces. Feces were then stored at –20oC, then thawed and dried in a forced air oven at 60oC for determination of dry matter percentages. The following fecal consistency scale was followed for visual scoring for consistency by a two-person panel (48).
SCORE #1: Runny liquid consistency, splatters on impact, spreads readily.
SCORE #2 Loose consistency yet may pile slightly, may splatter and/or spread moderately upon impact and settling.
SCORE #3 Soft, yet firm consistency, not hard, piles but spreads slightly upon impact and settling.
SCORE #4 Dry and hard, dry appearance and original form is not distorted on impact and settling.
On day 9 and 10 of each period, rumen samples from four fistulated Holsteins were collected at 0900h just prior to offering fresh feed and at 1530h for measurement of volatile fatty acids (VFA). Grab samples were obtained from the rumens of the fistulated Holsteins, combined, and strained through four layers of cheesecloth. One ml of 25% phosphoric acid and one ml of 30 mM 4- methylvaleric internal std. were added to five ml of strained rumen fluid and stored at –20 oC for later analysis of VFA. Samples were thawed, centrifuged, filtered, and molar proportions of volatile fatty acids were estimated by gas chromatography (HP 5890 Series II gas chromatograph, Agilent Technologies, Wilmington, DE). On d 9 or 10 of each period, pH measurements (VXR digital Model 2000, Orion Research, Inc.) were obtained at 0, 2, 4, 6, and 8 h post feeding (0830h, 1130h, 1330h, 1530h, and 1780h) in the similar locations as grab samples in the rumen.
Dry matter of saliva-contaminated sodium bentonite and sodium bicarbonate was determined by drying in a forced air oven at 60 oC to a constant weight. Intake of both bentonite and bicarb could then be more accurately determined.

Chemical analysis

Acid detergent fiber (ADF) and neutral detergent fiber (NDF) of corn and alfalfa silages was determined using the method of Goering and Van Soest to remove starch interference (38). NDF analysis of corn grain and soybean meal was determined using the procedure outlined by Van Soest (93). Crude protein was determined by nitrogen analysis for corn and alfalfa silages, corn meal, and soybean meal using macro-Kjeldahl procedure (68). Mineral analyses of K and S in corn silage and high moisture corn, and K, S, and Na in alfalfa silage were analyzed using inductively coupled plasma atomic emission spectroscopy (SpectroFlame Modula Tabletop ICP with autosampler Type FTMOA85D, Spectro Analytical Instruments, Inc., Fitchburg, MA).

Statistical Analyses

All data were analyzed using Proc Mixed in SAS.
The model for all 25 cows was:
Y= u + Gi + Bj + Fk + (BF)jk + P(k)l + Tm + (BT)jm + (FT)k m + (BFT)jkm + C(ij)n
+ (TC)(ij)mn + Eijklmn
Where:
Y= dependent variable: urine pH, urine specific gravity, blood protein, blood PCV, fecal pH, fecal score, fecal dry matter %, milk fat %, milk protein %, milk urea nitrogen, milk production (kg), fat-corrected milk, dry matter intake, intake/bodyweight %,
u= overall population mean,
Gi = effect of group (i=treatment sequence 1 vs. 2),
Bj = effect of breed (j=1, 2: Jersey, Holstein),
Fk= effect of free-choice option (k=1,2: no free-choice, with free-choice),
(BF)jk = interaction of breed and free-choice option,
P(k)l = effect of period nested within free-choice option (l= 1…4),
Tm= effect of diet (m=1,2: diet 1, diet 2),
(BT)jm= interaction of breed with treatment diet,
(FT)km= interaction of free-choice option with treatment diet,
(BFT)jkm = interaction of breed with free-choice options with treatment diet,
C(ij)n = effect of cow nested within group and breed (n=8 for Jerseys, n=17 for Holsteins),
(TC)(ij)mn= effect of cow by treatment diet, and Eijklmn = error.
The model for the 4 fistulated Holstein cows was :
Y = u + Gi + Fj + P(k)l + Tl + (FT)jl + Sm + (TS)lm + (FS)jm + (FTS)jlm + C(i)n
+ (TC)(i)ln + Eijklmn
Where:
Y= dependent variable: rumen pH, rumen VFA percentages,
u= overall population mean,
Gi = effect of group (i=treatment sequence 1 vs. 2),
Fj= effect of free-choice option (j=1,2: no free-choice, with free-choice), P(k)l = effect of period nested within free-choice option (k= 1…4),
Tl = effect of treatment diet (l=1,2: diet 1, diet 2),
(FT)jl = interaction of free-choice options with treatment diet,
Sm= effect of sampling time (m= 1…5 for rumen pH: 0, 2, 4, 6, and 8 hours post-feeding,
m= 1-2 for VFAs: 0 and 6 hours post-feeding),
(TS)lm= interaction of treatment diet with sampling time, (FS)jm= interaction of free-choice options with sampling time,
(FTS)jlm = interaction of free-choice options with treatment diet with sampling time, C(i)n = effect of cow nested within group (n= 2),
(TC)(i)ln = interaction of cow with treatment diet nested within group, and Eijklmn = error.
Due to illness, one Jersey was replaced in period 1 and a spare Jersey was used in
analysis. This same Jersey had to be replaced in period 2 because of habitual feeding
from calan doors which were not her own. One non- fistulated Holstein was replaced in period 2 because of illness and the spare Holstein was used in analysis.

RESULTS AND DISCUSSION

Data for both multiparous and primiparous cows were pooled for analysis due to scarce number of cows in some combinations of breed, treatment, and parity. Tables 2 and 4 contain results without and with 1.2% sodium bicarbonate in the total mixed ration. In addition, results without and with free-choice options are also listed in Table 2 and 4. Results are in Tables 3 and 5 for all 4 dietary regimes (D1-NFC, D2-NFC, D1-WFC, D2-WFC). For the four diet regimes, Tukey tests were used to determine if means were different.
Dry matter intake, milk production, and milk composition Numerous studies have observed effects of sodium bicarbonate in the ration on intake and production parameters. Most have studied exclusively Holsteins fed diets with high concentrate ratios, typically 60% concentrate with corn silage as the base forage. Most positive results have been from studies that fed corn silage rather than grass or alfalfa silage as forage. Because the forages fed in this experiment were unusually low in fiber, a lower amount of grain was fed which may account for some differences with other experiments. The grains fed, however, were corn meal and high moisture corn, that contain rapidly fermentable starch. Holsteins and Jerseys responded differently to treatments and free-choice options, and when appropriate, will be discussed separately.

Holsteins

Effects of diet on dry matter intake are presented in Tables 2 & 3. Holsteins consumed more feed DM when sodium bicarbonate was added to the ration (25.4 vs. 24.2 kg/d, Table 2). In addition, DMI as a percentage of bodyweight was greater (4.14 vs. 3.98 %).
This is in agreement with other studies concluding force- fed sodium bicarbonate has a positive influence on intake (27, 88). In contrast, other studies have concluded sodium bicarbonate had no influence on intake (29, 31, 49). Free-choice options did not affect intake. Greatest differences in intakes were recorded when Holsteins consumed D1-NFC (24.1, Table 3) relative to D2-NFC (25.5) and D2-WFC (25.3). Milk production increased approximately 1 kg/d or more when sodium bicarbonate was added to the ration (Tables 2 and 3). It is possible that this production response was due to both increased intake and digestibility of the ration although digestion trials were not conducted in this experiment. This production response agrees with numerous studies (27, 49, 69, 88) although a majority of trials have reported no significant differences in milk production (24, 28, 31, 34, 49, 68, 94). Fat percentage was greater when free-choice options were available (3.87 vs. 3.76, Table 2). Although free-choice options of sodium bentonite and sodium bicarbonate have not been reported to affect fat test percentage, the increase of milk fat in other experiments that included sodium bicarbonate in the diet is well documented (25, 28, 39, 69). However, it has also been documented that sodium bicarbonate does not affect milk fat percentages (24, 50, 94). One study found an increase in milk protein percent when sodium bicarbonate was fed (88). Our results are in agreement with a majority of trials that found no changes in milk protein (24, 34, 68). There were no differences in milk production or components between the four dietary regimes (Table 3). Although free-choice options increased milk fat percentage, fat-corrected milk (FCM) did not significantly change between diet regimes and was not affected by diet or free-choice options. Fat-corrected milk (FCM) has been shown to increase with force- fed sodium bicarbonate (28, 51, 77); however, some studies show no differences (29, 34).

Jerseys

Unlike Holsteins, Jerseys did not consume more dry matter with force-fed sodium bicarbonate. Jerseys had increased DM consumption (kg/d and percentage of bodyweight) when free-choice options were offered (20.4 kg vs. 19.4 kg and 4.78 vs. 4.56%, Table 2). Jerseys displayed no differences in milk production, milk protein, or fat percentage, or FCM when sodium bicarbonate was force- fed. FCM increased approximately 2.36 kg when free-choice options were available (Table 2). This corresponds to 3.0 mcal extra energy needed for milk production. However, DMI increased only 0.96 kg/d, which allowed a 1.63 mcal/d increase in energy. This may indicate more efficient digestion when free-choice options were available, however, periods were short (10 d) and not designed to show long term production changes. There were no differences in FCM between the four dietary regimes (Table 3). Jerseys had lower MUN levels when sodium bicarbonate was force-fed (14.79 vs. 15.68, Table 2) yet had higher levels when allowed free-choice options (15.74 vs. 14.73). The highest MUN level was observed with cows consuming D1-WFC (16.04, Table 3) but was different only from D2-NFC (14.14). It is not known why MUN levels in Jerseys varied, but consumption of sodium bentonite may be a factor in increasing the amount of protein escaping microbial digestion in the rumen (14, 55).

Acid-base status

Tables 4 and 5 contain results of urinary, fecal, and rumen pH analysis.
Blood pH and implications on urine pH
Blood pH is maintained within a narrow range except under extreme conditions. Changes in blood acid-base status may be related to several factors. These may include bicarbonate secretion in saliva, abomasal acid secretion, and varied rates of acid utilization and absorption from the rumen (8, 29). If acid is not metabolized it must eventually be excreted by the kidneys. Significant changes in acid excretion can take place without significant changes in blood acid-base balance (28). Hence, blood pH, pCO2, and HCO3- were not measured in this experiment. It has been shown that if acidosis occurs in early lactation it can be compensated for by urinary excretion. Small changes in acid levels can be detected easily in renal excretions (28). Because changes in acid load can be detected through urinary excretion, net acid urinary excretion is a more sensitive measure of changes in acid-base balance (78, 79).

Dietary Cation-Anion Difference (DCAD)

According to the 2001 NRC (Nutrient Requirements of Dairy Cattle) and analysis through the Virginia Tech Forage Laboratory, mineral concentrations in corn and alfalfa silages, high moisture corn, ground corn, and soybean meal, our diets contained a (DCAD) of +17.4 mEq/100g of dry matter for diet 1 and +30.8 mEq/100g DM for diet 2 (Table 1) (63). A diet with a high positive DCAD will cause a mild metabolic alkalosis resulting in a reduction of plasma bicarbonate, increased urinary acid excretion, and decreased urinary pH (91). DCAD is calculated by the equation (10):
DCAD = mEq (Na++K+) – (Cl- +S-)/100ml

Urine pH

Urine pH is not well-documented in experiments with rumen buffers but some do report observations. Erdman et al. found sodium bicarbonate and magnesium oxide supplementation had no influence on urine pH (27). However, Ghorbani et al. found an increase in urine pH when sodium bicarbonate or sodium sesquicarbonate were force-fed (34). Both experiments fed corn silage with 60% concentrate. Differences in the two studies may be due to average days in milk (DIM) being different where Erdman studied cows immediately postpartum and Ghorbani studied cows with an average of 180 DIM. In our study, Holsteins had higher urine pH when sodium bicarbonate was force- fed in the ration (8.28 vs. 8.22, Table 4). In Jerseys, higher urine pH was observed when allowed free-choice options (8.28 and 8.22). Jerseys receiving no buffering agent (D1-NFC) had the lowest urine pH (8.20, Table 5). Both Holsteins and Jerseys had alkaline pH values averaging above 8.0. Ruminants tend to excrete alkaline urine except when diets high in concentrates are fed. Most hydrogen ions are excreted in the NH3 form in ruminants consuming high concentrate diets (78). This has been demonstrated in sheep and calves when urine pH is below 8.0 (78, 79). When pH is above 8.0, HCO3- appears to be the main ion involved in net acid excretion of lactating cows (79).

TABLE OF CONTENTS
TITLE
ABSTRACT
ACKNOWLEDGMENTS
LIST OF TABLES
LIST OF FIGURES
INTRODUCTION
REVIEW OF LITERATURE
I. Acid-base balance in dairy cattle
II. Rumen mineral buffers…
III. Free-choice consumption
IV. Salt and sodium intake
MATERIALS AND METHODS
I. Animals
II. Diets
III. Experimental design
IV. Measurements and sampling
V. Chemical analysis
VI. Statistical analysis
RESULTS AND DISCUSSION
I. Dry matter intake, milk production, and parameters
A. Holsteins
B. Jerseys
II. Acid-base status.
A. Blood pH and implications on urine pH
B. Dietary cation-anion difference (DCAD)
C. Urine pH
D. Fecal pH
E. Rumen pH
III. Rumen volatile fatty acids (VFA)
IV. Blood parameters
V. Urine specific gravity
VI. Fecal Score and Dry Matter
VII. Free-choice intake of sodium bentonite and sodium bicarbonate.
VIII. Interactions
A. Holsteins
B. Jerseys
IX. Cost Effectiveness
SUMMARY
CONCLUSIONS
REFERENCES
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