Partial Replacement of Forage with Nonforage Fiber Sources in Lactating Cow Diets. II. Digestion and Rumen Function

Partial Replacement of Forage with Nonforage Fiber Sources in Lactating Cow Diets. II. Digestion and Rumen Function M. N. Pereira1 and L. E. Armentano Department of Dairy Science University of Wisconsin Madison 53706

ABSTRACT

INTRODUCTION

Replacement of forage with cereal byproducts may be a viable alternative for feeding dairy cows. The objective of this experiment was to evaluate total tract digestion and rumen fermentation profile when diets were formulated to contain low-forage neutral detergent fiber (NDF) (12.6% forage NDF, 18.8% total NDF), adequate NDF from forages (20% forage NDF, 24.4% total NDF) or low-forage NDF with high levels of NDF from cereal byproducts (12.7% forage NDF, 35.1% total NDF). Sodium bicarbonate (0.8% of dry matter) was factorialized over these diets. Total tract apparent digestibilities of organic matter (OM) and carbohydrates were determined in 73 Holsteins. Eight rumen-cannulated cows were used concurrently to evaluate rumen fermentation profile and in situ degradation of forages. Bicarbonate did not increase NDF or OM digestibility, but increased intake of digestible OM. Rumen fermentation parameters were determined by dietary alfalfa NDF content. Adding alfalfa NDF to the low-forage, high-starch diet increased in situ degradation of forage NDF more than adding byproduct NDF. However, increased ruminal forage NDF degradability was not reflected in greater total tract NDF digestibility. Replacement of dietary starch with NDF from byproducts decreased OM digestibility, but energy intake was similar across diets due to increased intake. (Key words: digestibility, byproducts, dairy cows, buffer)

Forages usually are the major source of fiber in dairy rations. However, diets formulated with high quantity of nonforage NDF, and forage NDF (FNDF) content below current recommendations (NRC, 1989), may be desirable when forages are in short supply, have low nutritive value, or have a high nutrient price relative to other feeds sources. More NDF must be added from nonforage fiber sources (NFFS) feeds than from forage to achieve the same increase in fat test (Swain and Armentano, 1994). However, the NDF from most NFFS do not stimulate chewing activity as effectively as FNDF (Clark and Armentano, 1993, 1994, 1997; Depies and Armentano; 1995, Swain and Armentano, 1994), with the notable exception of whole cottonseed (Clark and Armentano, 1993). Decreased chewing activity when NFFS replace forage can decrease the flow of salivary buffer to the rumen, decreasing rumen pH and NDF degradation (Grant and Mertens, 1992). Chewing activity and rumen pH of lactating cows decreased when soyhulls replaced 42% of dietary forage in a 59% forage diet and total NDF was increased from 28 to 34% of diet DM (Weidner and Grant, 1994). Feeding sodium bicarbonate may be useful in increasing total tract NDF digestibility when cereal NFFS are a major component of low-forage diets. Sodium bicarbonate supplementation increased rumen pH at 12 h postfeeding and tended to increase total tract NDF digestibility when lactating cows were offered diets containing 38% NDF and 20% corn silage, 15% alfalfa, and 20% corn gluten feed (Firkins et al., 1991). Excessive starch fermentation in the rumen may depress fiber degradation through decreased pH (Mould et al., 1983), but starch may also decrease NDF fermentation independently of rumen pH (Grant and Mertens, 1992). Diets formulated with high-NDF levels from NFFS have lower starch than diets formulated to provide equal effective NDF from forage, therefore the direct negative effect of starch on fiber digestion should be less for these high-NDF diets. Ruminal NDF digestibility was equal for diets of similar total NDF content formulated by partial replacement of forage with NFFS (Cunningham et al., 1993; Feng et al., 1993; Sarwar et al., 1991; Van Vuuren

Abbreviation key: FDNF = forage NDF, kd = fractional rate of degradation, LFHN = low-forage, highNDF diet, LFMN = low-forage, medium-NDF diet, NC = negative control diet, NFFS = nonforage fiber sources, PC = positive control diet.

Received February 14, 2000. Accepted June 27, 2000. Corresponding author: L. E. Armentano; e-mail: learment@ facstaff.wisc.edu. 1 Present address: Universidade Federal de Lavras, Departamento de Zootecnia, Lavras, 37200-000, M.G., Brazil. 2000 J Dairy Sci 83:2876–2887

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et al., 1993). As NFFS have lower undigestible NDF fraction compared to forages (Bhatti and Firkins, 1995), the amount of potentially degradable NDF leaving the rumen may be greater when NFFS replace forage (Pantoja et al., 1994). In diets of low forage and high NDF content, large intestine fermentation may assume increased importance compared with ruminal fermentation. The objective of this experiment was to determine total tract DM and carbohydrate digestibility of diets formulated to contain low NDF, or added NDF provided by forages or cereal NFFS. The strategy for balancing these rations and the performance of the cows is reported in Pereira et al. (1999). The impact of feeding sodium bicarbonate upon total tract apparent digestibility was also evaluated. The effect of changing the level and source of fiber on rumen fermentation profile and concurrent in situ degradation of corn silage and alfalfa silage was determined. The proportional importance of ruminal and large intestine diet fermentation was evaluated indirectly to indicate factors limiting total tract NDF digestibility. MATERIALS AND METHODS Continuous Trial Total tract digestibility of diets varying in carbohydrate content was determined in 73 individually fed midlactation Holsteins. A low-forage, high-NDF (LFHN) diet was formulated to contain similar concentration of FNDF as a high-starch negative control diet (NC). These diets were compared with a higher-forage, positive-control (PC) diet. Diet LFHN was formulated with a mixture of wheat middlings, corn gluten feed, and brewers grain (Table 1). Sodium bicarbonate (Church and Dwight Co., Inc., Princeton, NJ) at 0.8% of diet DM was factorialized over diets NC, LFHN, and PC to form six treatments. Diets were offered once a day as a TMR in amounts adequate to allow 10% refusal of the feed offered. Cows were assigned to the experiment in six groups; the first group entered the experiment in September of 1993 and the last in May of 1994. Within group, cows were blocked by days in lactation and randomly assigned within blocks to one of the six treatments. Fecal sampling for total tract apparent digestibility determination occurred during d 66 to 69 following abrupt change to and continued feeding of a treatment. During the period of fecal sampling, daily aliquots of refusals from each cow, and a daily sample of corn silage, alfalfa silage, and concentrates used in diets LFHN, PC, and NC, were collected. Four-day composites were formed by combining equal amounts of each feed on an as-fed basis. Forage and refusal composites

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were dried at 57°C for 48 h, ground through a 1-mm screen with a Wiley Mill (model 4, Thomas Scientific, Philadelphia, PA), and then a 300-g subsample was dried at 57°C for 48 h to determine DM. The DM of each concentrate composite was determined by drying a 300-g sample ground through a 1-mm screen at 57°C for 48 h. The amount of feed DM offered to each cow per day was calculated from the daily as-fed weights of corn silage, alfalfa silage, and concentrate multiplied by their respective composite DM content. A TMR composite sample for diets NC, LFHN, and PC was formed for each one of the six fecal sampling periods by mixing dried and ground amounts of alfalfa silage, corn silage, and concentrate composites in proportion to their concentration in the offered TMR DM. The ash, NDF, and starch plus free glucose content of feces, formed TMR composite samples, and refusals were determined as described in Pereira et al. (1999). Fecal grab samples were collected at 4-h intervals during 4 d, starting 1 h later each day. A fecal composite for each cow was made based on equal fresh weights and dried at 57°C for 48 h, ground through a 1-mm screen, and a 300-g subsample dried again at 57°C for 48 h. Fecal production was determined using acid insoluble ash as an internal marker (Van Keulen and Young, 1977). Dried and ground samples of feces (5 g), diets (8 g), and refusals (8 g) were combusted for 8 h at 450°C and subsequently boiled for 15 min in 100 ml of 2 N HCl. The intake of marker was calculated by determining the concentration of marker in the TMR composite samples and in the refusal composites from each cow. Fecal purine excretion (g/d) was determined to estimate the relative amount of fermentation occurring in the lower tract. The purine assay was based on the procedures described by Zinn and Owens (1986) and Aharoni and Tagari (1991). Acetic acid (0.1 M) was used to dilute the sample after hydrolysis with 70% perchloric acid. Ammonium phosphate monobasic (8 ml, 0.2 M) and ammonium phosphate dibasic (1 ml, 0.2 M) diluted the filtrate (0.5 ml) and the silver nitrate (0.5 ml, 0.4 M). Standards were prepared by serial 1:2 dilution of a 0.0125 M guanine solution (Aldrich Chemical Company, Inc., Milwaukee, WI). Absorbance was measured at 254 nm. Rumen fluid samples obtained on d 54 were used to correlate rumen pH and VFA concentration to total tract NDF digestibility, and fecal purine excretion per kilogram of digestible OM intake was determined during d 66 to 69. Samples were obtained by percutaneous aspiration of ventral rumen contents (Garrett et al., 1999). Sampling time was 242 ± 31 (mean ± SD) minutes postfeeding. Cows were sampled in random sequence within block. Journal of Dairy Science Vol. 83, No. 12, 2000

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PEREIRA AND ARMENTANO Table 1. Feed ingredient and nutrient composition (% of DM) of negative control (NC), low-forage, highNDF (LFHN), and positive control (PC) diets during the period of fecal sampling for determination of total tract apparent digestibility. No bicarbonate

Alfalfa silage Corn silage Ground dry shelled corn Soybean meal, 49% CP Corn gluten meal Meat and bone meal Blood meal Wheat middlings Brewers grain Corn gluten feed Minerals and vitamins Sodium bicarbonate1 CP Forage NDF NDF Starch + free glucose

Bicarbonate

NC

LFHN

PC

NC

LFHN

PC

17.0 14.4 45.4 17.8 1.3 1.7 1.0 ... ... ... 1.4 ... 18.9 12.6 18.9 43.6

17.0 14.5 14.8 0.1 1.4 2.8 1.0 15.5 14.5 17.0 1.4 ... 19.2 12.7 34.6 29.3

34.2 14.6 34.8 5.5 6.5 2.5 1.0 ... ... ... 0.9 ... 19.0 20.0 24.4 35.2

17.0 14.4 44.9 17.3 1.4 1.7 1.0 ... ... ... 1.5 0.8 19.1 12.6 18.7 43.4

17.0 14.5 14.0 0.1 1.4 2.8 1.0 15.5 14.5 17.0 1.4 0.8 18.9 12.7 34.4 29.3

34.2 14.6 34.1 5.4 6.5 2.5 1.0 ... ... ... 0.9 0.8 19.3 19.9 24.3 35.1

1

Church and Dwight Co., Inc., Princeton, NJ.

Latin Square Trial Eight rumen-cannulated Holsteins were used to evaluate ruminal digestion parameters of diets. The design of the experiment was a replicated 4 × 4 latin square with 35-d periods. Three diets were formulated to be equal to diets NC, LFHN, and PC of the continuous trial. An additional diet, low-forage, medium-NDF (LFMN) had the same content of FNDF as diets NC and LFHN. The concentrate for diet LFMN was formulated by mixing equal parts of concentrates NC and LFHN, on an as-fed basis (Table 2). Starting on d 31 of each period, an in situ evaluation of diet effect on forage ruminal degradation was performed. Pecap Polyester bags (10 × 15 cm), monofilament, 51-µm pore size (Tetko Inc., Briarcliff Manor,

NY) were filled with approximately 5 g of DM of either corn silage or alfalfa silage. Wet forages were ground with dry ice through a 5-mm screen. Both corn silage and alfalfa silage were incubated in all cows in all periods. All bags were inserted into the cows just before feeding (10 a.m.) and removed after 2, 4, 6, 12, 24, 48 and 72 h of incubation. Bags were soaked in warm water before being inserted into the cows and held in the rumen by a laundry bag containing a 500-g weight and attached to the rumen cannula plug by a 70-cm cord. Upon removal from the rumen, bags were immediately soaked in ice water and subsequently washed in warm tap water until a clear water flow was observed. Bags incubated for 48 h or less were frozen. After the 72-h

Table 2. Feed ingredient and nutrient composition (% of DM) of negative control (NC), low-forage, mediumNDF (LFMN), low-forage, high-NDF (LFHN), and positive control (PC) diets during the Latin square trial. Feed

NC

LFMN

LFHN

PC

Alfalfa silage Corn silage Ground dry shelled corn Soybean meal, 49% CP Corn gluten meal Meat and bone meal Blood meal Wheat middlings Brewers grain Corn gluten feed Minerals and vitamins CP Forage NDF NDF Starch + free glucose

17.8 14.3 45.6 16.8 1.6 1.7 1.0 ... ... ... 1.3 19.0 12.6 19.5 43.5

17.9 14.4 31.4 8.7 1.3 2.3 1.0 7.0 7.0 7.7 1.3 19.0 12.6 27.5 34.7

18.0 14.4 17.1 0.4 1.2 2.8 1.0 14.1 14.1 15.6 1.2 19.0 12.7 35.7 26.0

36.3 14.5 33.7 4.4 6.7 2.5 1.0 ... ... ... 0.8 18.8 20.1 25.2 33.2

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bags were removed from the rumen and washed in warm tap water, all bags for that period were thawed and cleaned in a washing machine. Zero-hour bags were washed in warm tap water and with the other incubated bags in the same washing machine cycle. Empty bags, feeds, and bags with residue were dried at 57°C for 48 h for dry weight determination. Parameters of ruminal degradation were determined by cow within period. Forage DM and NDF as a percentage of the initial bag content were used to determine fractions A and C, and the fractional rate of degradation (kd) of the slowly degradable fraction (B = 100 − A − C). Fraction C was considered to be the residue of the 72-h bag as a percentage of the initial bag content. Fraction A, the water soluble fraction and small particle loss, was estimated as 100 minus the bag residue at time 0 expressed as a percentage of the initial bag content. The kd of B was determined by linearly regressing, over time, the natural logarithm of bag residues as a percentage of the initial bag content after subtracting fraction C from that number. Time 0 bag residue as a proportion of the initial bag content was averaged by period and the mean value used for all cows within period to calculate the kd of fraction B. Feed 24-h degradation was calculated from 24-h bag DM and NDF disappearance expressed as a percentage of the initial bag content. Samples of rumen fluid were obtained on d 31 of each period at 0, 2, 4, 6, 9, 12, 15, 18, and 21 h postfeeding. A 150-ml glass cup was capped with the hand and opened into the ventral rumen. Samples were immediately strained through two layers of cheesecloth and the pH measured with a Corning 150 ion analyzer with a general purpose combination electrode. Trichloroacetic acid (50%, 1 ml) was added to 50 ml of strained rumen fluid, and the sample was frozen for ammonia determination (Chaney and Marbach, 1962). Strained rumen fluid (1.5 ml) was immediately centrifuged for 15 s at 15,000 × g using a Beckman Microfuge E. The supernatant (1 ml) was pipetted into a flask containing 43 µl of oxalic acid (0.72 M) and stored on ice until it could be frozen. After thawing, the sample was centrifuged at 1300 × g for 15 min at 5°C using a Beckman GPR centrifuge. Metaphosphoric acid (25%, 80 µl) was added to 400 µl of the supernatant. Samples were analyzed for VFA by GLC with a Perkin Elmer AutoSystem gas chromatograph equipped with a Supelco glass column packed with G.P. 10% SP − 1200/1% H3PO4 on 80/100 chromosorb W AW, at a column temperature of 115°C. Hourly values for rumen VFA (mM), ammonia (mg/ dl), and pH were balanced for sampling intervals, and the mean value was determined by the sum divided by 24. Minimum pH is the lowest value observed in any one of the nine daily sampling times. Time of rumen

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pH below 6 (min/d) was calculated for each cow within period, assuming a linear change in rumen pH within each sampling interval. Blood samples were obtained on d 33 of each period for insulin determination. Samples were obtained between 10 and 12 h postfeeding. Blood was centrifuged at 850 × g for 20 min at 20°C and serum stored at −20°C. Insulin was analyzed by radioimminoassay with a CoatA-Count kit (Diagnostic Products Corp., Los Angeles, CA) as modified by Studer et al. (1993). On the same day milk samples from the afternoon milking were obtained to determine the concentration of C18:1 trans and cis fatty acids in milk fat. Long-chain fatty acids were quantified by GLC as described by Gaynor et al. (1994). Statistical Analysis Continuous trial. Total tract apparent digestibility and fecal purine data were analyzed using the GLM procedure of SAS (1985) with the following model: Yijkl = µ + Gi + Bj(i) + Fk + Bil + Dbikl + Eijkl where: = = = = =

overall mean, group effect (i = 1,2,3,4,5,6), block within group effect (j = 1 to 13), fiber effect (k = NC, LFHN, PC), bicarbonate effect (l = bicarbonate, no bicarbonate), FBikl = interaction of fiber and bicarbonate, and Eijkl = residual error, assumed independently and identically distributed in a normal distribution with mean zero and variance σ2. µ Gi Bj(i) Fk Bil

Degrees of freedom for diet were partitioned into two single degree of freedom orthogonal contrasts: LFHN versus NC and LFHN versus PC. Within each diet, measurements of total tract NDF digestibility, rumen VFA concentration, and fecal purine over digestible OM intake from each cow were regressed against ruminal pH. Latin square trial. Rumen fluid variables, plasma insulin, and milk C18:1 trans fatty acids were analyzed using the GLM procedure of SAS (1985) with the following model: Yijkl = µ + Si + Cj(i) + Pk + Dl + Eijkl where: µ = overall mean, Si = square effect (i = 1,2), Journal of Dairy Science Vol. 83, No. 12, 2000

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Figure 1. Rumen acetate to propionate ratio as a function of time post feeding. Data from eight cows fed negative control (NC, 䊏), lowforage, medium-NDF (LFMN, ▼), low-forage, high-NDF (LFHN, ▲), and positive control (PC, 䊉) diets during two concurrent 35-d period 4 × 4 latin squares. Cows were fed at 10 a.m. Interaction of diet and time postfeeding: P = 0.26.

Cj(i) Pk Dl Eijkl

= = = =

cow within square effect (j = 1 to 8), period effect (k = 1,2,3,4), diet effect (l = NC, LFMN, LFHN, PC), and residual error, assumed independently and identically distributed in a normal distribution with mean zero and variance σ2.

Degrees of freedom for diet were partitioned into three single degree of freedom orthogonal contrasts: LFHN versus PC, linear effect of NFFS NDF addition (NC vs. LFHN), and quadratic effect of NFFS NDF addition (NC + LFN vs. LFMN). The significance of a period × square interaction was evaluated, and since it was not significant (P > 0.10), it was not included in the final model. Rumen data over time for pH, A/P, total VFA, and ammonia were analyzed using the repeated measures approach of the MIXED procedure of SAS (Littell et al., 1996) to obtain least square means and standard errors for Figures 1 to 4. The structure of covariance with the largest value for the Akaike’s information criterion was used (Littell et al., 1996). The model was similar to the one used for the analysis of ruminal parameters and degradation data (whole plot) with the addition of a time postfeeding effect and the interaction of time and diet. Parameters of ruminal degradation of corn silage and alfalfa silage DM and NDF were analyzed as a split plot design using the GLM procedure of SAS (1985) with the following model: Journal of Dairy Science Vol. 83, No. 12, 2000

Figure 2. Rumen pH as a function of time post feeding. Data from eight cows fed negative control (NC, 䊏), low-forage, medium-NDF (LFMN, ▼), low-forage, high-NDF (LFHN, ▲), and positive control (PC, 䊉) diets during two concurrent 35-d period 4 × 4 latin squares. Cows were fed at 10 a.m. Interaction of diet and time postfeeding: P = 0.26.

Yijkl = µ + Si + Cj(i) + Pk + Dl + SCPDijkl + Fm + FDlm + Eijkl where: µ = overall mean; Si = square effect (i = 1,2); Cj(i) = cow within square effect (j = 1 to 8);

Figure 3. Rumen VFA as a function of time post feeding. Data from eight cows fed negative control (NC, 䊏), low-forage, mediumNDF (LFMN, ▼), low-forage, high-NDF (LFHN, ▲), and positive control (PC, 䊉) diets during two concurrent 35-d period 4 × 4 latin squares. Cows were fed at 10 a.m. Interaction of diet and time postfeeding: P = 0.16.

REPLACEMENT OF FORAGE WITH BYPRODUCTS

Pk = period effect (k = 1,2,3,4); Dl = diet effect (l = NC, LFMN, LFHN, PC); SCPDijkl = interaction of square, cow, period, and diet (mean square used as the error term to test diet effect); Fm = forage effect (m = corn silage, alfalfa silage); FDlm = interaction of forage and diet; and Eijkl = residual error, assumed independently and identically distributed in a normal distribution with mean zero and variance σ2. Correlation analyses across diets were performed between parameters of ruminal degradation of the NDF in alfalfa and corn silage and rumen fluid variables. RESULTS AND DISCUSSION Starch plus free glucose digestibility was uniformly high, although it was slightly greater for diet LFHN compared with NC (Table 3). The NDF digestibility coefficient was lower than the starch coefficient, and was not affected by diets with different ratios of nonforage NDF to FNDF (Table 3). As a result, the DM and OM digestibility was determined by the NDF contents of the diets, being lowest for the LFHN, intermediate for the PC, and highest for the NC diet. The NDF in NFFS is sometimes assumed to be of higher digestibility than that in forage. Indeed, total tract NDF digestibility is usually increased when soyhulls NDF is added to the

Figure 4. Rumen ammonia as a function of time post feeding. Data from eight cows fed negative control (NC, 䊏), low-forage, medium-NDF (LFMN, ▼), low-forage, high-NDF (LFHN, ▲), and positive control (PC, 䊉) diets during two concurrent 35-d period 4 × 4 latin squares. Cows were fed at 10 a.m. Interaction of diet and time postfeeding: P = 0.06.

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diet in replacement of forage or concentrate (Cunningham et al., 1993, Sarwar et al., 1991, 1992). However, the NDF in cereal NFFS seems to be less digestible than soyhulls NDF (Bernard and McNeill, 1991). The present experiments do not support the concept that cereal NFFS are sources of highly digestible fiber, at least when included in diets containing low-forage levels. Digestible OM intake throughout the trial was calculated by multiplying weekly OM intake (Pereira et al., 1999) by the OM digestibility coefficient (Table 3). Weekly intake values were adjusted for the digestible OM intake before the period of treatment application as described in Pereira et al. (1999). Digestible OM intake throughout the continuous trial were (kg/d): 16.6 for diet NC, 16.2 for diet LFHN, and 16.5 for diet PC (P = 0.59). Sodium bicarbonate tended to increase (P = 0.07) digestible OM intake from 16.2 to 16.8 kg/d, with no interaction among diets (P = 0.24). The highest DMI (Pereira et al., 1999) compensated for the lowest digestibility of the LFHN diet (Table 3), resulting in equal digestible OM intake of all diets throughout the 112-d continuous trial. There was no evidence for increased rumen fill when NFFS NDF was added to diet NC (Pereira et al., 1999). The similarity of digestible OM intake across diets, combined with least milk fat yield with diet NC and greatest with diet PC, is consistent with the changes in BCS observed during the continuous trial (Pereira et al., 1999). Increased DMI also compensated for a decrease in dietary energy content when beet pulp plus rice completely replaced barley at 45 and 65% of diet DM (Thomas et al., 1986). The highest intakes obtained with the highNDF diet and the equality in energy intake across diets suggest that intake of energy was regulated, not intake of NDF. The mean fecal purine excretion across diets was 14.2 g/d (Table 3), equivalent to 94 millimoles of daily purine excretion. The mean ratio of rumen bacterial CP (g) to bacterial purine (millimol) was reported to be 3.99 (Vagnoni and Broderick, 1997). Assuming the same ratio of bacterial CP to purine in rumen and large intestine bacteria, the estimated daily excretion of bacterial CP synthesized in the large intestine would be 375 g in this experiment. Based on measured OM digestibility (Table 3), the mean dietary NEL content of these diets would be 1.74 Mcal/kg of DM (Moe and Tyrrell, 1976) and daily NEL intake would be 38.6 Mcal. The estimated flow of bacterial CP out of the rumen, based on the daily NEL intake (NRC, 1989), would be 2566 g. Large intestine synthesis of bacterial CP was estimated to represent 12.8% of the sum of intestinal plus rumen bacterial CP flows in this experiment, similar to the proportional contribution of large intestine VFA proJournal of Dairy Science Vol. 83, No. 12, 2000

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Table 3. Intake during the period of fecal sampling, digestibility coefficients, and fecal composition for cows fed negative control (NC), lowforage, high-NDF (LFHN), and positive control (PC) diets; supplemented with 0.8% of sodium bicarbonate (BC) or not (NB). NC NB

LFHN NB

PC NB

NC BC

LFHN BC

PC BC

SEM

Fiber1

Bicarbonate

NC vs. LFHN

PC vs. LFHN

Fiber by bicarbonate

DMI, kg/d DMD,2 % OMD,2 % SGD,2 % NDFD,2 % Fecal SG,2 % of DM Fecal NDF,2 % of DM

21.8 79.1 80.3 95.2 54.4 9.4 40.8

22.6 69.9 71.8 96.2 53.7 3.6 53.0

19.8 76.2 77.7 96.4 55.5 5.4 44.5

23.0 77.6 78.8 95.0 51.3 9.3 40.8

23.7 71.2 73.1 96.5 54.8 3.4 53.8

21.6 74.4 75.8 95.1 52.0 6.6 44.8

1.0 1.3 1.3 0.6 2.3 0.6 0.6

0.03 <0.001 <0.001 0.11 0.82 <0.001 <0.001

0.09 0.54 0.50 0.44 0.33 0.52 0.42

P 0.44 <0.001 <0.001 0.04 0.53 <0.001 <0.001

0.01 <0.001 <0.001 0.29 0.82 <0.001 <0.001

0.93 0.37 0.38 0.37 0.51 0.40 0.81

Fecal purine Concentration, mg/g Excretion, g/d Excretion/DOMI,2 g/kg

2.61 12.5 0.76

2.03 14.3 0.96

2.67 13.2 0.91

2.85 14.9 0.89

1.92 13.5 0.86

2.89 16.7 1.07

0.09 1.2 0.07

<0.001 0.54 0.09

0.12 0.09 0.25

<0.001 0.85 0.23

<0.001 0.38 0.27

0.08 0.19 0.12

Fiber = Main fiber source effect (NV vs. LFHN vs. PC). DMD = DM digestibility coefficient, OMD = OM digestibility coefficient, SGD = starch + free glucose digestibility coefficient, NDFD = NDF digestibility coefficient, Fecal SG = fecal starch + free glucose content, DOMI = digestible OM intake. 1 2

duction to total VFA production in ruminants (Siciliano-Jones and Murphy, 1989). Purine fecal excretion did not differ among diets, resulting in the lowest purine concentration in diet LFHN, the diet with greatest fecal DM output. Purine fecal excretion expressed as a proportion of digestible OM intake (Table 3) was used as an index of large intestine fermentation per unit of digestible energy intake. There was no evidence for a shift of diet fermentation from rumen to large intestine as a result of the dietary treatments applied in this study (Table 3). Sodium bicarbonate did not increase DM or NDF digestibility, irrespective of dietary carbohydrate nature (Table 3). The absence of positive response in rumen pH to feeding of bicarbonate (Pereira et al., 1999) may explain the lack of correlation between buffer and NDF

digestibility in this experiment. Lack of response of NDF digestibility to bicarbonate was also observed by Firkins and Eastridge (1992) in low-forage, corn silagebased diets containing soyhulls. Wagner et al. (1993) similarly observed no increase in NDF digestibility when sodium bicarbonate-supplemented diets containing 40% corn silage and 15% wheat middlings. The rumen fermentation profile was determined by the level of forage in the diet (Table 4). The addition of NFFS NDF had no effect upon the ratio of acetate to propionate. Low-forage diets had a lower acetate to propionate ratio than did diet PC throughout the day (Figure 1). Rumen pH in the low-forage diets tended to be lower than in diet PC, even though the total VFA concentration was similar for all diets (Table 4). The addition of FNDF to diet NC increased eating and rumi-

Table 4. Rumen fluid parameters based on nine rumen samples collected throughout a 24-h period. Data from eight cows fed negative control (NC), low-forage, medium-NDF (LFMN), low-forage, high-NDF (LFHN), and positive control (PC) diets during two concurrent 35d period 4 × 4 latin squares.

Acetate, % Propionate, % Butyrate, % Valerate, % Isobutyrate, % Methyl-butyrate,2 % Total VFA, mM Acetate/Propionate pH Mean pH Minimum Time below pH 6, min NH3 Mean, mg/dl

NC

LFMN

LFHN

PC

SEM

Diet

Linear1

Quadratic1

LFHN vs. PC

51.0 29.4 12.7 3.7 0.83 2.4 122.1 1.78 6.02 5.53 382 17.2

53.1 27.9 13.0 3.2 0.83 2.0 121.2 1.98 6.07 5.58 334 16.3

53.4 28.4 12.6 3.1 0.74 1.8 115.9 1.92 6.06 5.61 340 15.4

58.2 23.0 13.1 2.5 0.95 2.2 115.0 2.65 6.19 5.71 247 14.0

0.95 1.33 0.55 0.22 0.05 0.11 2.85 0.13 0.04 0.05 36.4 1.30

<0.001 0.01 0.90 <0.01 0.04 <0.01 0.22 <0.001 0.08 0.12 0.09 0.38

0.92 0.26 0.37 0.03 0.08 <0.001 0.14 0.47 0.51 0.29 0.43 0.33

P 0.22 0.79 0.44 0.47 0.38 0.69 0.55 0.43 0.51 0.82 0.56 0.99

0.04 0.04 0.67 0.08 0.01 0.04 0.83 <0.001 0.06 0.18 0.09 0.48

1

Linear and quadratic effects of NFFS NDF addition. Sum of 3-methyl and 2-methyl butyrate which coelute.

2

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REPLACEMENT OF FORAGE WITH BYPRODUCTS Table 5. Degradation of corn silage and alfalfa silage DM in situ. Data from eight cows fed negative control (NC), low-forage, medium-NDF (LFMN), low-forage, high-NDF (LFHN), and positive control (PC) diets during two concurrent 35-d period 4 × 4 latin squares.

Corn silage NC LFMN LFHN PC Alfalfa silage NC LFMN LFHN PC SEM

A1

B2

C3

kd4

24DEG5

52.4 52.4 52.4 52.4

20.8 23.2 23.0 28.3

26.8 24.3 24.5 19.2

3.21 3.24 3.83 3.40

59.8 62.6 62.1 65.3

41.2 41.2 41.2 41.2 0.23

33.1 33.6 33.8 34.6 0.84

25.7 25.2 25.1 24.2 0.92

5.40 5.84 6.02 6.79 0.37

64.6 66.5 67.5 68.4 0.71

<0.001 <0.001 <0.01 0.11 0.32 <0.01

<0.001 0.05 0.02 0.11 0.32 <0.01

0.11 <0.001 0.35 0.08 0.80 0.61

0.03 <0.001 0.38 0.08 0.38 0.16

P Diet effect Forage effect Diet*Forage Linear6 Quadratic6 LFHN vs. PC

<0.001

A = 0-h bag DM disappearance (% of DM). B = 100 − A − C (% of DM). 3 C = 72-h bag DM residue (% of DM). 4 kd = Fractional rate of degradation of fraction B (%/h). 5 24DEG = 24-h bag DM disappearance (% of DM). 6 Linear and quadratic effects of NFFS NDF addition. 1 2

Table 6. Degradation of corn silage and alfalfa silage NDF in situ. Data from eight cows fed negative control (NC), low-forage, medium-NDF (LFMN), low-forage, high-NDF (LFHN), and positive control (PC) diets during two concurrent 35-d period 4 × 4 latin squares. A1

B2

C3

Corn silage NC LFMN LFHN PC

6.8 6.8 6.8 6.8

38.7 44.7 44.3 55.2

54.5 48.5 48.9 38.0

2.82 3.01 3.44 3.15

18.0 23.8 22.4 30.2

Alfalfa silage NC LFMN LFHN PC SEM

7.6 7.6 7.6 7.6 0.59

43.5 45.0 45.6 47.3 1.77

48.9 47.4 46.8 45.1 1.81

4.49 5.16 4.94 6.53 0.43

32.9 36.3 38.0 39.8 1.27

<0.001 0.78 0.01 0.05 0.26 <0.01

P <0.001 0.73 0.01 0.05 0.26 <0.01

0.02 <0.001 0.14 0.12 0.59 0.07

0.02 <0.001 0.09 0.10 0.36 0.09

Diet effect Forage effect Diet*Forage Linear6 Quadratic6 LFHN vs. PC

0.35

kd4

24DEG5

A = 0-h bag NDF disappearance (% of NDF). B = 100 − A − C (% of NDF). 3 C = 72-h bag NDF residue (% of NDF). 4 kd = Fractional rate of degradation of fraction B (%/h). 5 24DEG = 24-h bag NDF disappearance (% of NDF). 6 Linear and quadratic effects of NFFS NDF addition. 1 2

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PEREIRA AND ARMENTANO

centration 21 h postfeeding. Differences in protein sources as well as differences in carbohydrate fermentation and eating patterns all may contribute to temporal changes in ruminal ammonia levels. Diet NC had a greater proportion of soybean meal protein compared with alfalfa and NFFS protein in the other diets. Estimates of dietary soluble protein were calculated (Net Carbohydrate and Protein System, 1995) to be 25% of CP in diet NC, and 30 and 34% of CP in diets PC and LFHN, respectively. The minimum ruminal concentration of ammonia, valerate, and methyl-butyrate observed throughout the day were statistically (P < 0.01) higher for the NC diet (data not shown), possibly reflecting more constant levels of protein deamination. For the forages incubated in situ, NDF content as a percentage of DM was 41.3% for alfalfa silage and 38.6% for corn silage. The content of DM was 54.6% for alfalfa

Figure 5. Positive control diet. Regressions of total tract NDF digestibility and fecal purine excretion per intake of digestible OM against ruminal pH. The best fit response lines are: NDF digestibility = 95.55 − 6.77 pH, r2 = 0.06. Fecal purine/DOMI = 0.27 + 0.12 pH, r2 = 0.03. P for regressions are greater than 0.23.

nation time per unit of DMI, while NFFS NDF addition had no effect (Pereira et al., 1999), suggesting that increased saliva buffer flow may be involved in the rumen pH response. However, throughout the day an inverse relationship between rumen pH and VFA concentration was observed for all diets (Figures 2 and 3). Mean rumen ammonia level was unaffected by dietary carbohydrate nature within this range of diets (Table 4). Similarly, for cows grazing high quality ryegrass, ruminal ammonia levels were equally lowered by supplementation with starch or NFFS based concentrates (Van Vuuren et al., 1986). However, the temporal pattern of rumen ammonia tended to vary among diets in this experiment (P = 0.06 for the interaction of diet and time postfeeding, Figure 4). A second peak of ammonia was observed 6 h postfeeding for all diets but NC, and diet NC had the highest rumen ammonia conJournal of Dairy Science Vol. 83, No. 12, 2000

Figure 6. Negative control diet. Regressions of total tract NDF digestibility and fecal purine excretion per intake of digestible OM against ruminal pH. The best fit response lines are: NDF digestibility = −576.04 + 201.08 pH − 15.94 pH2, r2 = 0.24. Purine/DOMI = 17.53 − 5.31 pH + 0.42 pH2, r2 = 0.25. P for regressions are smaller than 0.01.

REPLACEMENT OF FORAGE WITH BYPRODUCTS

Figure 7. Low-forage, high-NDF diet. Regressions of total tract NDF digestibility and fecal purine excretion per intake of digestible OM against ruminal pH. The best fit response lines are: NDF digestibility = 140.48 − 14.58 pH, r2 = 0.37. Purine/DOMI = −1.34 + 0.38 pH, r2 = 0.29. P for regressions are smaller than 0.01.

silage and 39.2% for corn silage. The addition of NFFS NDF to diet NC decreased the size of the C fraction of forages, but a greater decrease in fraction C was observed when FNDF was added (Tables 5 and 6). Pantoja et al. (1994) similarly decreased the rumen undegradable NDF fraction of alfalfa by adding either forage or soyhulls to a basal 15% FNDF diet. However, in our experiment the size of alfalfa fractions B and C were less affected by negative associative effects on forage degradation than the B and C fractions of corn silage. The NDF and DM C fraction of alfalfa were greater than corn silage fractions only when animals were fed the high-forage, PC diet (Tables 5 and 6). The different degradation responses of forages to dietary carbohydrate nature may result from differences in plant morphology (Wilson, 1993) and type of bacteria associated with fiber digestion of grasses and legumes (Akin,

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1979). Different mechanisms for inhibition of forage degradation may prevail for each forage. The kd of forage NDF tended to increase when NDF was added to diet NC (Tables 5 and 6). Adding forage to diet NC tended to increase the kd of FNDF more than NFFS addition. Batajoo and Shaver (1994) similarly observed that incremental replacement of corn-based concentrates with NFFS resulted in a linear increase in the kd of alfalfa hay DM. The effect of dietary NDF source and level upon the kd of forages was not statistically as marked as the effect upon the size of the C fraction (Tables 5 and 6). The kd of corn silage was lower than alfalfa (Tables 5 and 6). This is in accordance with the lower kd of grasses compared to legumes (Van Soest, 1995). Decreased in situ ruminal FNDF digestibility in diet NC (Tables 5 and 6) was not reflected in decreased total tract NDF digestibility (Table 3). It is unlikely that total tract digestion of NDF was maintained by increased NDF fermentation in the large intestine as diet NC tended to have the lowest fecal purine excretion (Table 3). For diet NC, a longer ruminal retention of FNDF may have compensated for the decreased ruminal degradation. Dry matter intake, especially forage DM, was lowest in diet NC (Pereira et al., 1999). Because the mass of ruminal NDF was equal in diets NC and PC (Pereira et al., 1999), low-forage intake for diet NC probably indicates low fractional turnover of the ruminal forage pool. Decreased chewing activity in diet NC (Pereira et al., 1999), which may reflect decreased ruminal motility (Dado and Allen, 1995), may have caused this reduction of fractional passage rate of forages. Digestion variables were regressed against rumen pH to visualize factors determining NDF digestibility within each diet (Figures 5 to 7). There was no correlation of rumen pH with NDF digestibility or with fecal purine excretion within diet PC (Figure 5). The pH values in diet PC may have not been low enough (Mould et al., 1983) to inhibit ruminal fiber digestion extensively, resulting in the absence of correlation between rumen pH and NDF digestibility within this diet. Within diet NC, the lowest pH values were related to decreased NDF digestibility, increased rumen VFA concentration, and greater proportion of total OM digestion in the lower tract (Figure 6). The frequency of rumen pH below 5.5 was statistically higher for diets NC and LFHN than for diet PC (Pereira et al., 1999). Decreased ruminal and total tract NDF digestibility may have occurred within diet NC in animals experiencing extreme pH depression, possibly the result of high starch fermentation. Fiber digesting microorganisms may have been inhibited in these cases by the conjunct action of low pH and the predominance of Journal of Dairy Science Vol. 83, No. 12, 2000

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PEREIRA AND ARMENTANO Table 7. Serum insulin (µU/ml) and milk fat C18:1 trans and cis fatty acids (% of total fatty acids) during the wk 5 of the latin square trial. Data from eight cows fed negative control (NC), low-forage, medium-NDF (LFMN), low-forage, high-NDF (LFHN), and positive control (PC) diets. NC

LFMN

LFHN

PC

SEM

Diet

Linear1

Quadratic1

44.4 2.64 4.9 22.2

30.8 2.76 4.3 23.8

30.0 3.14 3.1 24.6

30.5 3.23 2.3 22.0

3.1 0.09 0.5 0.9

<0.01 <0.001 0.01 0.15

<0.01 <0.001 0.03 0.07

0.14 0.25 0.58 0.70

LFHN vs. PC

P Insulin Milk fat, % C18:1 Trans C18:1 Cis

0.74 0.47 0.31 0.06

1

Linear and quadratic effects of NFFS NDF addition.

starch as the major substrate for microbial growth (Grant and Mertens, 1992). These data are consistent with a greater proportion of lower tract fermentation, possibly due to increased flow of fermentable NDF to the large intestine. Increased intestinal fermentation apparently was not sufficient to compensate for the decreased ruminal NDF digestibility in these cases. Within diet LFHN (Figure 7), lowered rumen pH was related to increased NDF digestibility and decreased proportion of diet fermentation in the lower tract as indicated by purine excretion. Low pH apparently did not decrease NDF digestibility in diet LFHN (Figure 7), even though the frequency of low rumen pH was as prevalent as in diet NC (Pereira et al., 1999). Because VFA production on this diet is probably more dependent on NDF than on starch as a substrate, higher total tract NDF digestibility may reflect greater rumen NDF digestibility, resulting in increased rumen VFA and decreased rumen pH. Lowered ruminal retention time of NDF may be involved in depressions in NDF digestibility in diets high in NFFS (Bhatti and Firkins, 1995; Weidner and Grant, 1994). Ruminal contents of cows receiving diet LFHN were observed to be more homogeneous with a less distinct mat than in diet PC, possibly decreasing the retention time of small NDF particles. Cows fed NFFS with more rapid rumen turnover may have had decreased rumen VFA, increased pH, and increased the flow of fermentable NDF to the lower tract, resulting in higher fecal purine. Strategies for increasing NDF digestibility in low-forage, high-NDF diets need to consider the retention of small NDF particles in the rumen. Effects of the different carbohydrate feeding strategies on milk trans fatty acids and blood serum insulin and corresponding milk fat concentration (Table 7) support the theory that trans 18:1 fatty acid(s) are causally related to milk fat depression (Gaynor et al., 1994). Although insulin was elevated by the high-starch diet, which had the lowest milk fat concentration, the differences in milk fat concentration among the high-fiber diets were not accompanied by differences in insulin. This response supports both the observation that highJournal of Dairy Science Vol. 83, No. 12, 2000

starch diets increase insulin and decrease milk fat concentration (Jenny et al., 1974) but that the increased insulin is not the cause of the lowered milk fat concentration (Griinari et al., 1997). SUMMARY Sodium bicarbonate supplementation did not increase NDF or OM digestibility in high-energy diets fed to lactating cows, but it did increase intake of digestible OM. The addition of NDF to a low-forage, high-starch diet increased in situ degradation of forage NDF, and alfalfa NDF addition was more beneficial than adding NDF from NFFS. The increased ruminal degradation to added NDF was greater for corn silage than for alfalfa. However, increased ruminal forage NDF degradability was not reflected in greater total tract NDF digestibility across diets. The replacement of dietary starch with NFFS NDF decreased total tract OM digestibility, but energy intake was similar across diets. ACKNOWLEDGMENTS We gratefully acknowledge Rich Erdman for analysis of the trans-fatty acids in milk fat. We also appreciate the financial support of Church and Dwight Co. (Princeton, NJ) and the Brazilian Federal Agency for Post-graduate Education (CAPES). REFERENCES Aharoni, Y., and H. Tagari. 1991. Use of nitrogen-15 determinations of purine nitrogen fraction of digesta to define nitrogen metabolism traits in the rumen. J. Dairy Sci. 74:2540–2547. Akin, D. E. 1979. Microscopic evaluation of forage digestion by rumen microorganisms—A review. J. Anim. Sci. 48:701–710. Batajoo, K. K., and R. D. Shaver. 1994. Impact of nonfiber carbohydrate on intake, digestion, and milk production by dairy cows. J. Dairy Sci. 77:1580–1588. Bernard, J. K., and W. W. McNeill. 1991. Effect of high fiber energy supplements on nutrient digestibility and milk production of lactating dairy cows. J. Dairy Sci. 74:991–995. Bhatti, S. A., and J. L. Firkins. 1995. Kinetics of hydration and functional specific gravity of fibrous feed by-products. J. Anim. Sci. 73:1449–1458. Chaney, A. L., and E. P. Marbach. 1962. Modified reagents for determination of urea and ammonia. Clin. Chem. 8:130–132.

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