Ruminal Digestion and Microbial Utilization of Diets Varying in Type of Carbohydrate and Protein1,2 S. R. STOKES, W. H. HOOVER. T. K. MILLER. and R. BLAUWEIKEL Dhridon of Animal &d Veterinary Sciences West Virginia University Morgantown 26606 ABSTRACT
Three ruminally and duodenally cannulated, lactating Holstein cows were used in a 3 x 3 Latin square experiment to study the effects of differing levels of nonstructural carbohydrate and ckgradable intake protein on ruminal digestibility and microbial protein production. Three diets were formulated to contain 1) 38 and 13.2%,2) 31 and 11.8%, and 3) 24 and 9% nonstructural carbohydrate and degradable intake protein as percentages of the DM, respectively. Dry matter intakes were similar for all diets (21.9, 21.1, and 18.3 kg/d for diets 1, 2, and 3, respectively). Likewise, microbial efficiency, as estimated from purine analysis, was unaffected by diet and averaged 24 g of microbial N/kg of OM digested for all treatments. Ruminal digestion of OM averaged 66.6, 65.1, and 55.7% for diets 1, 2, and 3, respectively, resulting in lower microbial N flow per day for diet 3 (317, 333, and 202 g, respectively). Digestion of nmstructuralcarbohydrate and CP followed similar trends as did OM digestion, whereas NDF digestion remained similar across all diets. These results indicate that nonstructural carbohydrate greater than 24% and ruminally degradable protein greater than 9% of DM will enhance microbial protein flow from the rumen.
(Key words: nonstructural carbohydrate, degradable intake protein, rumen metabolism) Abbreviation key: CHO = carbohydrate, DAPA = diaminopimelic acid, DIP = degradable intake protein, EN = endogenous N, NSC = nonstructural carbohydrate, UIP = undegradable intake protein. INTRODUCTION
In order to meet the nutrient demands of lactating cattle, the flow of nutrients from rumen fermentation should be maximized before supplementing the diet with bypass sources of protein and energy. When feeding the rumen microbial population, efficiency of substrate utilization involves a strong interaction between the carbohydrate and protein fractions in the ration (22, 23). A positive response in microbial metabolism to increasing dietary N or carbohydrate has been observed in vitro (15, 16, 29); however, dietary fractions of protein and carbohydrate in current dairy rations need clearer definition with respect to ruminal digestion and microbial synthesis. Recently, several studies were conducted to observe the relationship between sources of nonstructural carbohydrate (NSC)and degradable intake protein (DIP) in the rumen of the lactating cow. Herrera-Saldana and Huber (11) observed increased milk production in response to a diet formulated with rapidly degradable carbohydrate and protein (barley plus cottonseed meal) compared with less ruminally Received July 13, 1990. degradable diets of milo and brewers grains. In Accepted October 4,1990. 'Published with the approval of the director of the West a subsequent experiment fmm the same group, Virginia Agricultural awl Forestry Experiment Station as using the same diets with cannulated cows (lo), Scientific Article Number 2244. Supported by funds pro- the barley plus cottonseed meal diet was shown vided by the Hatch Act and by Agway Inc., Syracuse, NY. to promote greater microbial protein produc%he authom graterulry acLnowIedge the donation of tion. Others (4, 19) have reported no response the canola meal by CSP Food Ltd., Wiunipeg, Manitoh with appreciation extended to R. Hallock for arranging the in milk production to synchronizing ruminal donation and shipment. protein and carbohydrate degradation. McCar1991 J Dairy Sci 74:871-881
87 1
872
STOKES ET AL.
thy et al. (19) found that although ruminal N flow and OM digestion were greatest for barley-based diets supplemented with soybean meal, corn-based diets (a more slowly degraded starch source) supported higher levels of milk production. These responses were confounded by intakes that were significantly lower with barley-based diets and may have masked responses due to source of protein or carbohydrate. Macleod et al. (18) reported an increase in milk production in response to increasing the NSC content of the diet to 37% with no additional benefit from higher levels. Other studies with lactating cows in which NSC was >30% of the ration have been conducted to investigate responses to varying protein sources (35, 37, 38). Increasing the DIP was generally associated with increased microbial N flow from the m e n and microbial efficiency. Reports were not found for in vivo studies in which both NSC and DIP levels were varied; therefore, review of the literature did not reveal a clear relationship among NSC, DIP, microbial growth, and animal performance. The objective of this experiment was to examine ruminal digestion and microbial m e tabolism as influenced by type of carbohydrate and protein in the ration. MATERIALS AND METHODS Animal Management and Diet Formulation
TABLE 1. Ingredient composition and analysis of basal ration. Item Ingredient Corn silage
Grass hay Ground shelled corn Corn gluten meal Dried brewers grains Canola meal F i meal
Urea Limestone Agmate' Nutrient content 8 NEL? Mcal/kg
NDF
(46 of D M 26.8 26.8 26.0 4.1 8.1 5.2 1.4 .4 .7 .5 17.7 1.61
33.9
lS. 22% K, 18%; M g , 11%. '~stimated NRC requirements (21).
ived from a previous study in continuous culture (33) and were used to estimate daily microbial protein yield as a function of DM digestibility and microbial efficiency. For each diet, undegradable intake protein (UIP) was supplied in the amount necessary to complement the predicted microbial protein flow and provide a total flow of 3.5 kgld of CP from the rumen. Diets were balanced for basic nutrient requirements (NE=, CP, and minerals) determined by the NRC (21) for cows producing 36 kgld of milk (average production of the three cows). Urea was added to diets 1 and 2 to achieve soluble protein levels of approximately 30% of CP (Table 2). Urea was not included in diet 3 because this diet was formulated to contain sources of energy and protein with greater ruminal bypass contents; thus, the objective with this diet was not to meet the animal's requirements with ruminal fermentation products. Diets were formulated to meet minimum NRC requirements of NEL; however, as a result of greater inclusion of concentrate feedstuffs in diets 1 and 2, the NEL of these diets averaged 6% greater than in diet 3. Sodium bicarbonate was added to all diets at 1.5% of DM to aid in maintaining rumen pH.
Seventy days prior to expected calving date, three multiparous Holstein cows were duodenally cannulated (14) with a rigid, T-type cannula (ANKOM, Spencerport, NY). Cows were maintained on grass hay at ad libitum intake until 10 d prior to calving when ground corn was supplemented gradually to a maximum of 1% of BW. After calving, cows were fed a basal diet (Table 1) until 36 d postpartum, at which time the experiment was initiated. One week postpartum, each cow was fitted with a rumen cannula (10cm inner diameter, Bar Diamond, Inc., Parma, ID). Ingredient and chemical composition of experimental diets is listed in Table 2. Diets were designed to have three levels of NSC (38, 31, and 24% of DM) with DIP included to give Experimental Procedure NSC:DIP ratios of 2.7 (13.7, 11.8, and 9.0%of and Sample Collection DM). Prediction equations (Table 3) based on Diets were assigned in a Latin square dedietary NSC and DE' concentrations were der- sign. Experimental periods were 17 d with d l Journal of Dairy Science Vol. 74, No. 3, 1991
RUMEN DIGESTION OF F'RO'IXINS AND CARBOHYDRATES
873
to 12 for adaptation to diet and d 13 to 17 for sample collection. Cows were fed their respective diets at ad libitum intake twice daily (WOO Diet1 and 2100 h) in a total mixed ration. Hay and Item 1 2 3 straw were ground in a tub grinder (John Deere, Ingredieas % East Moline, IL) (2.5cm screen) to facilitate 9.3 33.5 15.4 mixing. Diet samples were taken weekly and 13.0 21.2 28.6 2 s Wheat straw . . . . . . 15.4 composited frozen over the entire trial. Orts 41.6 165 Ground corn 18.9 were weighed daily, composited d 12 to 17, and Corn gluten meal . . . 6.0 15.7 frozen. Beginning on d 3 of each period, 100 g Wheat middlings . . . 18.0 . . . of Yb-labeled diet (prewash4 in acidified waCanola meal 25.6 8.5 ... ter, labeled with 50 g of YbCl3 and 10 L of Fishmeal ... 3.0 2.2 Urea .7 .3 . . . HzO/kg of diet, and dried at SOT) were dosed Megalac3 . . . . . . 2.3 intraruminally before each feeding as a digestiAgmate4 .2 .4 .4 bility marker. On d 13 to 16, 500 ml of duo~imestone~ 1.5 .8 .4 denal contents were collected at 12-h intervals, Chemical component advancing 3 h daily. Samples were composited m ~ McaVkg 6 1.70 1.71 1.61 and frozen for analyses. Ruminal digesta samCP,% 18.7 18.4 18.1 Soluble proteiq7 % CP 31.0 27.4 13.8 ples were collected on d 16 at 0 time (before Degradable protein,' % CP 73.3 64.4 49.9 feeding) and at 3, 6, 9, 12, 20, 22, and 24 h NDF, % 27.4 33.1 39.9 postfeeding. Each sample was strained immediNonstruchrral carbohydrate, % ately through four layers of cheesecloth, the pH 24.4 Enzyme amlysis 38.2 31.3 was determined, and a 200-ml aliquot was 25.4 calculated9 39.2 32.2 Fat," % 9.3 9.7 11.1 acidified and frozen. An additional 200 ml were composited in a vessel containing 4 ml of 'Sodium bicarbonate included at 1.5% of diet DM. 21ncluded a timothy, orchardgrass, and fescue mix; saturated mercuric chloride for microbial harvesting. Bacteria were harvested on d 17 after contained 9.3% protein and 64% NDF. 'Church and Dwight Co., Inc.: 82.5% fat (minimum). the 24-h collection according to the methods described by Smith and McAllan (31). Protozoa 4S, 22%; K, 18%; Mg, 11%. Included to meet a 1 0 1 were harvested from 1 L of strained rumen N:S ratio for each diet. fluid collected 3 h postfeeding. Prior to harvestbcluded to meet or exceed NRC requirements (21) and to maintain a 2:l C z P ratio for each diet. ing, protozoa were prepared and counted (1). The remaining strained fluid was transferred to % l u e s calculated from data of NRC (21). three 500-ml separatory funnels, each contain7Values calculated from data of Pox et al. (6). ing 150 ml of saline supplemented with 5 @. 'Values calculated from data of Reston (26). '[lOO - (CP + (NDF - NDF bound protein) + ash + of dextrose. After incubation at 39'C for 3 h, the protozoa were drawn off and washed three fat)]. times by centrifugation using saline, with the ''~mform-metaano1 extraction. final wash in distilled H 2 0 . On d 16 and 17, TABLE 2. Ingx&ents and chemical composition of expdmental diets on a DM basis.
s3
TABLE 3. In vitro prediction equations used to calculate microbial yield. DM Digestion (%; corrected for microbial matter) = 83.7 - (2.788 NSC) + (2.358 DIP) + (.037 NSC2) Microbial efficiency (B of m/kg digested DM) = -13.5 + (.876 NSC) + (2.672 DIP) - (.011 NSC2) - (.055 D S ) where: NSC = Dietary nonstructural carbohydrate level, percentage of DM. DIP = Dietary degradable intake protein level, percentage of DM. MN = Microbial N.
Journal of Dahy Science Vol. 74, No. 3, 1991
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STOKES ET AL.
TABLE 4. ~ntalreand milk production means for diets1
~~~~
DW
~~
Wd
DMI, % of B W
~~
~~
A B C X A B C -
X
Milk yield, kgld
A
B C
x
22.1 22.9 20.8 21.9 3.7 4.2 3.2 3.7 44.7 43.4 24.6 37.5
21.9 22.3 19.1 21.1 4.0 4.1 3.1 3.7 40.2 39.3 23.5 34.3
16.8 18.3 19.7 18.3 2.7 3.3 3.2 3.1 30.9 34.4 26.0 30.4
NS
.5
NS
.I
NS
1.3
'Diet 1 was formdated to contai0, as percenta%es of DM, 38% nonsln~chnalcarbohydrate (NSC) and 13.7% degradable intake protein PIP);diet 2, 31% NSC and 11.8% DP, diet 3, 24% NSC and 9% DIP. 2 o r t h o g o ~contrasts, w e NS (P > .IO).
cows were bled via jugular venipuncture 3 and 12 h postfeeding. Blood was centrifuged at 2000 x g for 20 min at 4'C. Serum was decanted and frozen until analysis. Cows were milked twice daily with weights recorded at each milking. The mean of the milk yields for d 10 to 17 were used to compare effects of diets.
microbial) and ammonia N in liquid samples (duodenal and m e n ) were determined by AOAC methods (3). Fat in dried diet, ort, and duodenal samples was extracted with chloroform:methanol (2: 1) using automated equipment (Tecator Inc., Hemdon, VA). In addition, Yb-labeled diets and duodenal samples were analyzed for Yb concentration by atomic absorption spectrophotometry following the proc e d w of Hart and Polan (9). Ytterbium conSample Analyses centration in the duodenal samples was used to Diets and orts were dried at 50'C and calculate DM flow to the duodenum and rumiground through a l-mm screen for analyses. nal digestion. Diaminopimelic acid P A P A ) Liquid duodenal samples were homogenized in and RNA concentrations in dried duodenal and two successive wet grinds using a Telanar SD- microbial samples were determined for calcula45 disperser ( T e h a r Co., Cincinnati, OH)first tions of microbial matter by methods of Webfor 8 min using a G450 generator, followed by ster (36) and Zinn and Owens (39), respectivea second grinding for 4 min with a G454 ly. Liquid m e n samples were analyzed for generator. Subsamples were lyophilized and VFA by gas chromatography (34). Serum samground through a 1-mm screen. Diets, orts, and ples were analyzed for urea N concentrations dried duodenal samples were analyzed for NDF photometrically using a S m i W e Diagnostic and ADF by methods of Goering and Van kit (Smith-Kline-Beclanan, Philadelphia, PA) Soest (8) with modification by Robertson and in which diacetylmonoxine was the colorimetVan Soest (27). The NSC content of each diet ric reagent. was determined by both enzyme analysis (30), modified to use ferricyanide as a colorimetric Statistical Analysis agent, and by difference calculations [lo0 Data were analyzed by ANOVA for a 3 x 3 (CP + (NDF - NDF bound protein) + ash + Latin square design using the general linear ether extract)]. Both methods gave similar models procedure of the SAS (32). Model was values; for the rest of this paper, NSC intakes blocked on cows and stage of lactation. Due to and digestibilities are calculated from values ration design, orthogonal contrasts were used to derived by enzyme determination. Total N and test for h e a r and quadratic responses to dOM in dried samples (diet, on, duodenal, and nal availability of carbohydrate and protein. Journal of Dairy Science Vol. 74, No. 3, 1991
875
RUMEN DIGESTION OF PROTEINS AND C A R B O H Y D ~ T E S
TABLE 5. Intakes* and digestibilities of OM, total carbohydrate, and carbohydrate fractions in response to diet2 Diet
SEM
1
2
3
contrast3
19.5
19.5
16.5
NS
"5
13.0 66.6
12.7 65.1
9.2 55.7
NS L
.8 1.4
14.3
14.3
10.5
NS
1.o
9.2 64.1
9.6 67.1
5.8 55.8
NS
NS
.8 4.5
8.8
7.3
4.6
L
.3
6.2 70.6
5.5 74.2
2.2 46.6
L NS
.4 5.9
55
6.9
6.6
NS
.5
2.9 54.0
3.9 565
3.6 55.5
NS
.4
NS
1.5
3.4
3.6
4.2
NS
.4
1.9 45.8
2.0 48.7
2.0 49.9
NS
-1
NS
3.0
OM
Intake. Wd Digested in the rumen4 kgld % OM Intake Total carbohydrate' Intake, kg/d Digested m the m e n kdd % CHO Intake Nonstructural carbohydrate Intake, kg/d Digested in the m e n kdd % NSC Intake NDF Intake, kg/d Digested in the rumen kgld Sb NDF Intake ADP Intake, kg/d Digested in the rumen kgld % ADP Intake
'Intakes adjusted for orts composition. 2Diet 1 was formulated to contain, as percentages of DM, 38% nonstNctural carbohydrate (NSC) and 13.7% degradable htake protein (DIP); diet 2, 31% NSC and 11.8% DP, diet 3, 24% NSC and 9% DIP. 3orthogonal contrasts where L = hear (P < .a) a ~ dNS (P > .IO). 4C0mtcd for microbial OM; calcnlated using RNA analysis of microbial matter. 5 ~ o t a CHO l = struchrnil + Nsc.
with an increase in milk production as did cows A and B, it should be noted that cow C produced similar milk yields throughout the last Intakes, Milk Production, two periods (diets 2 and 1, respectively) in and Dlgestlbllltles spite of the loss of a quarter to mastitis. Dry matter intakes and milk production maAverage intakes and digestibilities of OM ble 4) were not different among diets (P > .lo). and carbohydrate (CHO) fractions are preMilk production was greater with diet 1 > 2 > sented in Table 5. Intakes of OM were similar 3. Lack of statistical differences in intake and for all diets, but OM digested in the rumen milk yield appear due to cow C maintaining decreased as NSC and DIP decreased (P c .05). essentially constant consumption and milk pro- Diets were formulated to provide approxiduction irrespective of diet. Cow C received mately 65% of DM as total CHO, with the difdiets in the order of 3, 2, and 1; on d 12 of ference among diets 1,2, and 3 being in N D F period 2 (diet 2), she was diagnosed with acute NSC ratios, which were 4258, 51:49, and 62: mastitis, which resulted in the loss of a quarter. 36, respectively. Total CHO intake and ruminal To allow for this, period 2 for cow C was digestibility were not different among diets, extended until feed intake returned to normal but, due to diet composition, intake of NSC and was maintained (an additional week). Al- differed (P < .05) among diets. Nonstructural though cow C did not respond to diets l and 2 carbohydrate digestion (percentage of NSC inRESULTS AND DISCUSSION
Journal of Dairy Science Vol. 74, No. 3, 1991
876
STOKES ET AL.
TABLE 6. Ruminal VFA levels, ruminal pH, ruminal NH3 N, and serum
u r ~ aN
as affected by diet'
~~
Diet VFA, mM3
1
2
3
contras?
SEM
142.6
121.4
99.0
L
9.4
65.9 19.5 10.6 1.43 1.39
NS NS NS NS NS NS NS NS
.9
VFA, moVlOO mol
Acetate (A) Propionate (p) Butyrate Isobutyrate Isovalerate Valerate A:P PH3 NH3 N? mg/d senun urea N," mg/dl
56.7 26.9 12.0 1-46 1.35 1.59 2.40 5.9 21.2 19.8
59.9 22.8 12.7 1.31 1.45 1.57 2.66 6.3 15.0 23.4
1.24
3.39 6.6 8.0 23.7
L
Q
.8
.2 .6 .1
.2 .3 .2 1.6 .8
Diel 1 was formulated to contain, as percentages of DM, 38%nonStnrcIuralcarbohydrate (NSC) and 13.7%degradable intake protein PIP);diet 2, 31% NSC and 11.8% DIP; diet 3, 24% NSC and 9% DIP. 2onhogonal contrasts, where L and Q = linear and quadratic (P < .05), respectively, L = linear (P < .lo) and NS (P> .lo). 3Average of five sample times over 1 2 4 period. 4Average of four sample tima over 48-h period.
take) decreased with diet NSC level. Although diets 1 and 2 appeared to be similar, diet 2 did not deviate statistically from a linear line. Of the total CHO digested in the rumen, the NSC fraction accounted for 68% with diet 1, 59% with diet 2, and only 38% with diet 3. Although the major source of starch was similar in all diets (86, 70, and 75% of NSC from ground corn plus corn silage for diets 1, 2, and 3, respectively), diets 1 and 2 averaged 55% higher NSC digestibility compared with diet 3. Others have reported decreased starch digestion in lactation rations with low DIP (11). Ruminal digestion of NSC with diets 1 and 2 was higher than reported in the literature with diets containing >3Wo of DM as NSC and in which corn was the major source (19). Intakes of the fiber fractions (NDF and A D n were lower for diet 1 than for diets 2 and 3, but differences were NS (P > .lo). Digestibility coefficients for NDF and ADF were similar across all diets, averaging 55 and 48%, respectively. Of the total CHO digested in the rumen, the NDF fraction accounted for 32% with diet 1,41% with diet 2, and 62% with diet 3. It appears that when the ruminally digested carbohydrate fraction shifted from >50% NSC to >50% NDF, intake and OM digestion of the J o d of Dairy Science Vol. 74, No. 3, 1991
diets were reduced. The maintenance of fiber digestion (as a percentage of NDF intake) with diets high in NSC agrees with other reports (10, 11) but is in conflict with the results of McCarthy et al. (19), who noted a depression in NDF digestion as ruminal availability of starch inCreaSed. Volatlle Fatty Acld Productlon, Rumlnal Condltlons, Serum Urea Nltrogen, and Mlcroblal Composltlon
Concentrations of total ruminal VFA decreased (P < .lo) as ruminal availability of carbohydrate and protein decreased flable 6). Volatile fatty acid concentration with diet 1 was similar to literature values reported for diets containing starch levels greater than 30% of the ration and supplemented with ruminally degradable protein sources (19). The trend for change in individual molar proportions of VFA (Table 6) reflects the CHO composition of the diets. These differences, however, were NS and indicate that the changes in NDF:NSC ratios in this study did not markedly alter VFA ratios. The high concentration of total VFA in diet 1 is consistent with the low ruminal pH (Table 6) observed with this diet. Ruminal pH below 6 may have a detrimental effect on microbial
877
RUMEN DIGESTION OF PROTEINS AND CARBOHYDRATES
\
---_DIET
\
1
3
t
5.6 0
1
I
I
2
4
6
1
I
I
8 1 0 1 2 HOURS POST FEED
Figure 1. Ruminal pH fluctuations as affected by type of dietary carbohydrate and protein. Diet 1 was formulated to contain, as percentages of DM, 38%nonstruchnal carbohydrate (NSC) and 13.7%degradable intake protein (DE'); diet 2,31% NSC and 11.8%D P , and diet 3,2446 NSC and 9% DIP.
0
L---
0
3 6 9 HOURS POST FEED
12
Figure 2. Ruminal ammonia N concentrations as influenced by type of dietary carbohydrate and protein. Diet 1 was formulated to contain, as percentages of DM, 38% nonstructural carbohydrate (NSC) and 13.7% degradable intake protein (DIP); diet 2.31% NSC and 11.8%DIP, and diet 3, 24% NSC and 9% DIP.
suggesting that factors other than ruminal ammonia level affect blood urea N. reductions are cyclic and of short duration, the Microbial composition is presented in Table depression may be moderate (12). Ruminal pH 7. Bacterial content of N and ash were similar with diet 1 reached a low of 5.7 at 6 h after between diets 1 and 2 but were lower (P< .05) feeding but returned to >6 after t h i s time. and higher (P < .05), respectively, with diet 3. whereas ruminal pH with diets 2 and 3 re- Average bacterial compositions (percentage of mained between 6.2 and 6.8 throughout the day N and of ash) were similar to those reported in (Figure 1). Although the low pH observed with the literature (20), but the decrease in N and diet 1 appeared biologically significant, fiber increase in ash with diet 3 is in contrast to digestion (percentage of MDF intake) was not observations that diet type (forage versus coninhibited. centrate) did not alter microbial composition (7, Ammonia N concentrations (Table 6, Figure 24). Protozoal content of N and ash were not 2) decreased (P< .05) with increasing inclusion affected by diet, and average values were someof protein sources more resistant to ruminal what lower than literature reports (24). degradation. Diet 3 consistently resulted in the Microbial marker concentrations (RNA and lowest N H 3 N levels but remained above 5 mg/ D M A ) were similar across all diets. Bacterial dl even though this diet was formulated for and protozoal concentrations of RNA (11.21 degradable and soluble protein levels of 9.0 and and 7.53% of total N, respectively) were higher 2.5% of dietary DM, respectively. Serum urea than those reported by Arambel et al. (2) and N concentration was significantly lower (P < Ling and Buttery (17), whereas bacteria RNA .01) with diet 1 compared with diets 2 and 3, values were lower than those of Craig et al. (5). growth and cellulolytic activity; however, when
Journal of Dairy Science Vol. 74, No. 3, 1991
878
STOKES ET AL.
TABLE 7. Microbial composition and protozoal numbm as affected by diet.' ~~
I
Diet 2
3
contrast2
8.87 12.53 11.00 .350
8.87 11.74 11.13 .395
8.01 16.30 11.54 .390
Q
.12 .37
NS NS
.m .01
6.44 4.14 7.89
555 4.42 8.79
4.78 4.59 6.75
NS NS
.98 .23
NS
8.1
7.4
3.2
NS
Bacteria
%N %A& RNA N, % BN3 D N A N,4 % BN
Q
SEM
Protozoa
% N % Ash
RNA N, % PN' Protozoa count per ml nunen fluid (X ~
16, ~
~
~~
.w
15
~
'Diet I was formulated to contain, as percentages of DM, 38% nonstructural carbohydrate (NSC) and 13.7% degradable intake protein (DIP); diet 2, 31% NSC and 11.8% D E diet 3, 24% NSC and 9% DIP. 'Orthogonal contrasts, where Q = quadratic ( P < .05) and NS (P > .lo). 3Bacterial N. 4Diaminopimelic acid N. 5pr~t0zoalN.
Diaminopimelic acid concentrations of bacteria (.378% of total bacterial IV) were lower than those of Oluboboken et al. (24). Protozoal numbers (Table 7) were greater, but not significantly greater, with diets 1 and 2 compared with diet 3. The increase in protozoal population with diets 1 and 2 reflects the increase in entodiniomorphs noted with these diets (7.4 x 1 6 , 7.3 x 1 6 for diets 1 and 2, respectively) compared with diet 3 (3.1 x 16). Holotrich count remained unaffected by diet (average, 1.2 x 16). Nitrogen Metabo1ism
Nitrogen intake and degradation results are listed in Table 8. Although nonsignificant, intake of N with diets 1 and 2 averaged 15.5% greater than with diet 3 (P = .23). Due to intake and ruminal degradation differences, DIP levels were 12.5, 12.9, and 6.3% of DM, which were different from the original estimates made using NRC (21) values (13.7, 11.8, and 9.0%, respectively). Ruminal protein digestion was lower for diet 3 (P < .05) than for other diets and was lower than predicted (33.5 vs. 49.9%). Diets were designed to supply a 3.5 kg total CP flow to the duodenum, and flows of nitroge nous compounds to the duodenum were 3.4, 3.5, and 3.7 kg for diets 1,2, and 3, respective Journal of Dairy Science Vol. 74, No. 3, 1991
ly. Recovery of N at the duodenum (duodenal N/N intake) averaged 87% for diets 1 and 2, whereas recovery for diet 3 was 106%,reflecting the LJIP contribution to the duodenal protein supply. Nitrogen data in Table 8 were not corrected for endogenous contribution. Using an average 3.2 g of endogenous N (EN) per kilogram of duodenal DM flow (13, 28) gave estimates of 40.7, 41.1, and 41.2 s/d of EN flow for diets 1, 2, and 3, respectively. Accounting for these estimates of EN contribution, true dietary protein degradation values averaged 74.5, 74.6, and 41.2% for diets 1, 2, and 3, respectively. Microbial N production (Table 8) was determined using both DAPA and RNA concentrations. A criticism of the RNA technique is the common use of the RNA Ntotal N ratio of bacteria to estimate microbial N in duodenal contents containing both bacteria and protozoa. This is thought to give values underestimating actual microbial production because protozoa RNA N:total N ratios have been shown to be somewhat lower than those of bacteria (17). In this study, both bacteria and protozoal fractions were isolated and analyzed and an 80:20 weighted ratio was used in the RNA calculations (25). Although production of microbial N (RNA method) appeared similar between diets 1 and 2 (averaging 61% greater than diet 3).
879
RUMEN DIGESTION OF PROTEINS AND CARBOHYDRATES
TABLE 8. Intake and digestion of N and flow of nitrogenous compounds to the duodenum. Diet
N Intake? g/d Ruminal N digestiq4 % Intake Flow to the duodenum Total N, g NAN. g Microbial N, g RNA DAPA NA"? g RNA DAPA~ Microbial N g/kg OM truly digested RNA DAPA carbohydrate digested RNA DAPA
1
2
3
contrast2
sEh4
633
642
552
NS
34
69.0
68.0
33.5
Q
3.6
542 519
566 537
587 568
NS NS
30 30
317 367
333 376
202 266
L
10 38
201 15 1
204 161
366 302
L
NS
24.8 26.8
26.4 27.6
22.0 26.9
NS NS
35 5 40.6
35.0 39.7
345 45.7
NS NS
~~
23 8
Q
1.6 2.7 3.1 6.3 ~
~~
'Diet 1 was formulated to contain, as percentages of DM, 38% noostructaral carbohydrate (NSC) and 13.7% degradable intake protein (DIP); diet 2, 31% NSC and 11.8% DIP, diet 3, 24% NSC and 9% DIP. %rhogod contrasts where L and Q = limar and quadratic (P < -05). respectively, and NS (P > .lo). for orts composition. 3c0-ted 4Calculated using RNA to estimate microbial matter. %onammoria, nonmicrobial N. 6Diaminopimelic acid.
diet 2 did not deviate statistically from the linear response line. As a fraction of the total duodenal protein flow, microbial protein production supplied 59, 59, and 34% for diets 1,2, and 3, respectively. Microbial yields (RNA method) in this study were in the range of those repurted by McCarthy et al. (19) but lower than those observed by Herrera-Saldana et al. (10). Variation in microbial protein production may be explained partially by differences in methods of estimation, because Herrera-Saldana et al. (10) used an assumed nucleic acid content of microbial N, whereas McCarthy et al. (19), as in this study, measured microbial RNA. The greatest nonammonia, nonmicrobial N flow to the duodenum occurred with diet 3, which included protein sources having greater resistance to ruminal degradation. Microbial efficiencies (Table 8), expressed as grams of microbial N per kilogram of OM truly digested, did not differ among diets. Efficiencies in this study were lower than the
means of 38 and 32 g of microbial N/kg of OM truly digested observed with other studies (10, 19). It has been suggested that microbial yields should be expressed in terms of CHO rather than OM because CHO is the primary source of energy for microbial growth (22). Although microbial efficiencies expressed in this manner averaged 49% greater than those based on weight of OM digested, no differences due to diet type were noted. In summary, ruminal digestibility of OM, NSC, and CP was increased when NSC and DIP were >24 and 9% of DM, respectively; however, digestion was not further improved by increasing the level of NSC greater than 31% of the ration. Microbial protein synthesis responded similarly and was maximized with the NSC and DIP levels in diet 2 (31 and 12.9%of DM as NSC and DIP, respectively). Ruminal fermentation (as measured by OM digestion and VFA concentration) was enhanced with diets containing CHO and protein sources more Journal of Dairy Science Vol. 74, NO. 3, 1991
880
STOKES ET AL.
available in the rumen (diets 1 and 2 compared with diet 3). Microbial efficiency (expressed in terms of weights of OM truly digested or CHO digested) remained unaffected by diet, reflecting the proportionate reductions in both digestion and microbial protein synthesis with decreasing dietary NSC and DIP concentrations. This study was designed to explore the potential for manipulating the flow of fermentation end products and microbial protein from the rumen. Production of microbial N and levels of VFA in the rumen were enhanced by appropriate levels of NSC and DIP, which reduced the amount of bypass protein and fat needed to meet requirements for production. Diets formulated to contain, as percentages of DM, 31 or 39% NSC and 11.8 or 13.7% DIP supported greater microbial protein synthesis and VFA production than did a diet containing 25% NSC and 9% DIP. REFERENCES 1 Abe, M, and P. Kumeno. 1973. In vitro simulation of rumen fermentation: Apparatus and effects of dilution rate and continual dialysis on fermentation and protozoal populations. J. Anim. Sci. 36941. 2Arambel M. J., E. E. Bartley, S. M.Dennis, G. S. Ma,T. G. Naganja, D. E. Nuzback, D. 0. Riddell, A. D. Dayton, and S. J. Galitzer. 1987. Evaluation of several methods for estimating microbial nitrogen concentration in the rumen. Nu@. Rep. Int. 35%. 3Association of Ofticial Analytical Chemists. 1970. official methods of analysis. 11th ed. AOAC. Washinptq Dc. 4 Casper, D. P.,and D. J. Schingoethe. 1989. Lactational response of dairy cows to diets varying m ruminal solubilities of carbohydrate and crude protein. J. Dairy Sci. 72928. 5 Craig, W. M.,D. R Brown,G. A. Broddck and D. B. Ricker. 1987. Post-primdial changes of fluid- and particle-associated ruminal microorganisms. J. Anim. Sci. 65: 1042. 6 Fox,D. G., C. J. Sniffen, J. D. O'Connor. J. B.Russell. and P. J. Van SO&. 1986. The C ~ m e l lMt Carbohydrate and protein system for evaluating cattle diets. Comell Univ., Ithaca, NY. 7Fwchtenicht, J. E., and G. A. Broderick. 1987. Effect of inoculum prepantion and dietary energy on microbial numbem and rumen protein degradation activity. J. Dairy Sci. 701404. 8 Goeriog, H. K.,and P.J. Van Soest. 1970. Forage fiber analyses (apparatus, reagcnls, procedans, and some applications). Agric. Haedbook NO. 379. ARS-USDA,
Washington,Dc. 9% S. P., ami C. E. Polan. 1984. Simultaneous extraction and determination of ytterbium and cobalt ethylenediamineteeetate complex in feces. J. Dairy Journal of Dairy Science Vol. 74, No. 3, 1991
Sci. 67388. lOHerrera-Saldana, R., R. Gomez-Alarcon, M. Torabi, and J. T. Huber. 1990. Influence of synchronizing protein and starch d-on in the m e n on nutrient utilization and microbial protein synthesis. J. Dairy Sci. 73:142. 11Hemra-Saldaua, R, and J. T. Huber. 1989. Influence of varyiug protein and starch degradations on performamx of lactating cow. J. Dairy Sci. 72:1477. 12Hoover. W. H. 1986. chcmical factors involved in ruminal fiber westion. J. Dairy Sci. 692755. 13 Hvelplund, T., and J. h4adsea 1985. Amino acid passage to the small intestine in dairy cows compared with estimates of microbial protein and undegraded dietary protein from analysis on the feed. Acta Agric. Scand. Suppl. 2521. 14 Komarek, R J. 1981. Intestinal canudation of cattle and sheep with a t-shaped canuula designed for total digcsm collection without externalizing digesta flow.J. Anim. Sci. 53:7%. 15Lewis, D. 1962. The interrelationships of individual proteins and carbohydrates during fermentation in the rumen of the sheep. II. The fermentation of starch in the presence of starch or other substances containing nitrogen. J. Agric. Sci. (Camb.) 58:73. 16Lewis, D., and I. W. McDonald. 1958. 'Ihc interrelationships of individual proteins and carbohydrates during fermentation in the rumen of the sheep. I. The fermentation of casein in the presence of starch or other carbohydrate materials. J. Agric. Sci. (Camb.) 51: 108. 17Ling, J. R, and P. J. Buttery. 1978. The simultaneous use of ribonucleic acid, %, 2,&diaminopimelic acid as markers of microbial N entering the duodenum of sheep. Br. J. NUB. 39164. 18 Macl@ G. K.,D. G.Grieve, I. McMillaq and G. C. Smith. 1984. Effect of varying protein and energy densities in complete rations fed to cows in first lactation. J. Dairy Sci 67:1421. 19McCarthy, R D., Jr., T. H.Klusmeyer, J. L. Vicini, J. H.Clark, and D. R Nelson. 1989. Effects of source of protein and carbohydrate on ruminal fermentation and passage of nutrienfs to the small intestbe of lactaring cows. J. Dairy Sci. n 2 0 0 2 . 20Mary, R J., and A. B. McAuan. 1983. A comparison of the chemical composition of mixed bacteria harvested from the liquid and solid fractions of rumen digesta. Br. J. Nutr. 5Q701. 21Natiod Research Council. 1989. Nutrient requirements of dairy cattle. 6th rev. d.Natl. Acad. Sci., Washington, Dc. 22NoccL. I. E., and J. B. Russell. 1988. Protein and energy as an integrated system. Relationship of ruminal protein and carbohydrate availability to microbial synthesis and milk production. J. Dairy Sci. 71:2070. 23 Oldham, J. D. 1984. Protein-energy interrelationships in dairy cows. J. Dairy Sci. 67:1090. 24Oluboboken, 1. A., W.M. Craig, and W. A. Nipper. 1988. QzanrteriStic~Of protozoal and bactesial fractions from microorganisms associated with ruminal fluid or particles. J. Anim. Sci. 66:2701. 25pilgrim, A. F., F. V. Gray, R. A. Weller, and C. B. Belling. 1970. Synthesis of microbial protein in the sheep's rumen and the proportion of dietary nitrogen
RUMEN DIGESTION OF PROTEINS AND CARBOHYDRATES converted into microbial nitrogen. Br. J. Nutr. 24589. 26 Preston, R L. 1989. Typical composition of feeds for cattle, sheep. Feedstuffs 61(41):19. 27Robertson, J. B., and P. J. Van Soest. 1977. Dietary fiber estimation in concentrate feedstuffs. J. Anim. Sci. 45(Suppl. 2):254. 28 Rohr, K., P. Lebzien, H. Schafft, and E. Schulz. 1986. Prediction of duodenal flow of eon-ammonianitrogen and amino acid nitrogen in dairy cows. Livest. Prod. Sci. 1429. 29 Russell, J. B.,C. J. Sniffen, and P. J. Van Soest. 1983. Effect of carbohydrate limitation on degradation and utilization of casein by mixed rumen bacteria. J. D a q Sci. 66:763. 30Smith, D. 1969. Removing and analyzing total eonstructural carbohydrate from plant tissue. Wisconsin Agric. Exp. Stn. Res. Rep. 41:l. 31 Smith,R.H., and A. B. McAllan. 1974. Some factors influencing the chemical composition of mixed rumen bacteria. Br. J. Nutr. 31:27. 32 SASQ User’s Guide: Statistics. 1982. SAS Inst., Inc., Gary, NC. 33 Stokes, S. R.,W. H. Hoover, T. K. Miller, and R. P. Manski. 1991. Impact of carbohydrate and protein
881
level on bacterial metabolism in continuous culture. J. Dairy Sci. 74854. 34 Supelco Bulletin 749E. 1975. Supelco, Inc., Bellefonte, PA. 35 Waltz, D. M.,M. D. Stern, and D. J. J.Ug. 1989. Effect of ruminal protein degradation of blood meal and feather meal on the intestinal amino acid supply to the lactating cow. J. Dairy Sci. 721509. 36 Webster, P. M.,W. H. Hoover, and T. K. Miller. 1990. Determination of 2,64amhopimelic acid in biological materials using high performance liquid chromatography. Anim. Feed Sci. Technol. 30:ll. 37 Windscbitl, P. M.. and M. D. Stem. 1988. Evaluation of calcium l i g ~ ~ ~ ~ d f ~ ~ t e - tsoybean r e a t e dmeal as a s o w e of rumen protected protein for dairy cattle. J. Dairy Sci. 71:3310. 38Zerbi~i,E., C. E. Polan, and J. H. Herbeii 1988. & meal on Effecls of dietary soybean meal and f protein digesta in Holstein cows during early and midlactation. J. Dairy Sci. 71:1178. 39 Zinn, R A.. and F. N. Owens. 1982. A rapid procedure for microbial protein estimation. Page 26 in Protein reqimnents of cattle: symposium. F. N. Owens, ed. Oklahoma Agric. Exp. sta MP-109.
Journal of Dairy Science Vol. 74. No. 3, 1991
Three ruminally and duodenally cannulated, lactating Holstein cows were used in a 3 x 3 Latin square experiment to study the effects of differing levels of nonstructural carbohydrate and ckgradable intake protein on ruminal digestibility and microbial protein production. Three diets were formulated to contain 1) 38 and 13.2%,2) 31 and 11.8%, and 3) 24 and 9% nonstructural carbohydrate and degradable intake protein as percentages of the DM, respectively. Dry matter intakes were similar for all diets (21.9, 21.1, and 18.3 kg/d for diets 1, 2, and 3, respectively). Likewise, microbial efficiency, as estimated from purine analysis, was unaffected by diet and averaged 24 g of microbial N/kg of OM digested for all treatments. Ruminal digestion of OM averaged 66.6, 65.1, and 55.7% for diets 1, 2, and 3, respectively, resulting in lower microbial N flow per day for diet 3 (317, 333, and 202 g, respectively). Digestion of nmstructuralcarbohydrate and CP followed similar trends as did OM digestion, whereas NDF digestion remained similar across all diets. These results indicate that nonstructural carbohydrate greater than 24% and ruminally degradable protein greater than 9% of DM will enhance microbial protein flow from the rumen.
(Key words: nonstructural carbohydrate, degradable intake protein, rumen metabolism) Abbreviation key: CHO = carbohydrate, DAPA = diaminopimelic acid, DIP = degradable intake protein, EN = endogenous N, NSC = nonstructural carbohydrate, UIP = undegradable intake protein. INTRODUCTION
In order to meet the nutrient demands of lactating cattle, the flow of nutrients from rumen fermentation should be maximized before supplementing the diet with bypass sources of protein and energy. When feeding the rumen microbial population, efficiency of substrate utilization involves a strong interaction between the carbohydrate and protein fractions in the ration (22, 23). A positive response in microbial metabolism to increasing dietary N or carbohydrate has been observed in vitro (15, 16, 29); however, dietary fractions of protein and carbohydrate in current dairy rations need clearer definition with respect to ruminal digestion and microbial synthesis. Recently, several studies were conducted to observe the relationship between sources of nonstructural carbohydrate (NSC)and degradable intake protein (DIP) in the rumen of the lactating cow. Herrera-Saldana and Huber (11) observed increased milk production in response to a diet formulated with rapidly degradable carbohydrate and protein (barley plus cottonseed meal) compared with less ruminally Received July 13, 1990. degradable diets of milo and brewers grains. In Accepted October 4,1990. 'Published with the approval of the director of the West a subsequent experiment fmm the same group, Virginia Agricultural awl Forestry Experiment Station as using the same diets with cannulated cows (lo), Scientific Article Number 2244. Supported by funds pro- the barley plus cottonseed meal diet was shown vided by the Hatch Act and by Agway Inc., Syracuse, NY. to promote greater microbial protein produc%he authom graterulry acLnowIedge the donation of tion. Others (4, 19) have reported no response the canola meal by CSP Food Ltd., Wiunipeg, Manitoh with appreciation extended to R. Hallock for arranging the in milk production to synchronizing ruminal donation and shipment. protein and carbohydrate degradation. McCar1991 J Dairy Sci 74:871-881
87 1
872
STOKES ET AL.
thy et al. (19) found that although ruminal N flow and OM digestion were greatest for barley-based diets supplemented with soybean meal, corn-based diets (a more slowly degraded starch source) supported higher levels of milk production. These responses were confounded by intakes that were significantly lower with barley-based diets and may have masked responses due to source of protein or carbohydrate. Macleod et al. (18) reported an increase in milk production in response to increasing the NSC content of the diet to 37% with no additional benefit from higher levels. Other studies with lactating cows in which NSC was >30% of the ration have been conducted to investigate responses to varying protein sources (35, 37, 38). Increasing the DIP was generally associated with increased microbial N flow from the m e n and microbial efficiency. Reports were not found for in vivo studies in which both NSC and DIP levels were varied; therefore, review of the literature did not reveal a clear relationship among NSC, DIP, microbial growth, and animal performance. The objective of this experiment was to examine ruminal digestion and microbial m e tabolism as influenced by type of carbohydrate and protein in the ration. MATERIALS AND METHODS Animal Management and Diet Formulation
TABLE 1. Ingredient composition and analysis of basal ration. Item Ingredient Corn silage
Grass hay Ground shelled corn Corn gluten meal Dried brewers grains Canola meal F i meal
Urea Limestone Agmate' Nutrient content 8 NEL? Mcal/kg
NDF
(46 of D M 26.8 26.8 26.0 4.1 8.1 5.2 1.4 .4 .7 .5 17.7 1.61
33.9
lS. 22% K, 18%; M g , 11%. '~stimated NRC requirements (21).
ived from a previous study in continuous culture (33) and were used to estimate daily microbial protein yield as a function of DM digestibility and microbial efficiency. For each diet, undegradable intake protein (UIP) was supplied in the amount necessary to complement the predicted microbial protein flow and provide a total flow of 3.5 kgld of CP from the rumen. Diets were balanced for basic nutrient requirements (NE=, CP, and minerals) determined by the NRC (21) for cows producing 36 kgld of milk (average production of the three cows). Urea was added to diets 1 and 2 to achieve soluble protein levels of approximately 30% of CP (Table 2). Urea was not included in diet 3 because this diet was formulated to contain sources of energy and protein with greater ruminal bypass contents; thus, the objective with this diet was not to meet the animal's requirements with ruminal fermentation products. Diets were formulated to meet minimum NRC requirements of NEL; however, as a result of greater inclusion of concentrate feedstuffs in diets 1 and 2, the NEL of these diets averaged 6% greater than in diet 3. Sodium bicarbonate was added to all diets at 1.5% of DM to aid in maintaining rumen pH.
Seventy days prior to expected calving date, three multiparous Holstein cows were duodenally cannulated (14) with a rigid, T-type cannula (ANKOM, Spencerport, NY). Cows were maintained on grass hay at ad libitum intake until 10 d prior to calving when ground corn was supplemented gradually to a maximum of 1% of BW. After calving, cows were fed a basal diet (Table 1) until 36 d postpartum, at which time the experiment was initiated. One week postpartum, each cow was fitted with a rumen cannula (10cm inner diameter, Bar Diamond, Inc., Parma, ID). Ingredient and chemical composition of experimental diets is listed in Table 2. Diets were designed to have three levels of NSC (38, 31, and 24% of DM) with DIP included to give Experimental Procedure NSC:DIP ratios of 2.7 (13.7, 11.8, and 9.0%of and Sample Collection DM). Prediction equations (Table 3) based on Diets were assigned in a Latin square dedietary NSC and DE' concentrations were der- sign. Experimental periods were 17 d with d l Journal of Dairy Science Vol. 74, No. 3, 1991
RUMEN DIGESTION OF F'RO'IXINS AND CARBOHYDRATES
873
to 12 for adaptation to diet and d 13 to 17 for sample collection. Cows were fed their respective diets at ad libitum intake twice daily (WOO Diet1 and 2100 h) in a total mixed ration. Hay and Item 1 2 3 straw were ground in a tub grinder (John Deere, Ingredieas % East Moline, IL) (2.5cm screen) to facilitate 9.3 33.5 15.4 mixing. Diet samples were taken weekly and 13.0 21.2 28.6 2 s Wheat straw . . . . . . 15.4 composited frozen over the entire trial. Orts 41.6 165 Ground corn 18.9 were weighed daily, composited d 12 to 17, and Corn gluten meal . . . 6.0 15.7 frozen. Beginning on d 3 of each period, 100 g Wheat middlings . . . 18.0 . . . of Yb-labeled diet (prewash4 in acidified waCanola meal 25.6 8.5 ... ter, labeled with 50 g of YbCl3 and 10 L of Fishmeal ... 3.0 2.2 Urea .7 .3 . . . HzO/kg of diet, and dried at SOT) were dosed Megalac3 . . . . . . 2.3 intraruminally before each feeding as a digestiAgmate4 .2 .4 .4 bility marker. On d 13 to 16, 500 ml of duo~imestone~ 1.5 .8 .4 denal contents were collected at 12-h intervals, Chemical component advancing 3 h daily. Samples were composited m ~ McaVkg 6 1.70 1.71 1.61 and frozen for analyses. Ruminal digesta samCP,% 18.7 18.4 18.1 Soluble proteiq7 % CP 31.0 27.4 13.8 ples were collected on d 16 at 0 time (before Degradable protein,' % CP 73.3 64.4 49.9 feeding) and at 3, 6, 9, 12, 20, 22, and 24 h NDF, % 27.4 33.1 39.9 postfeeding. Each sample was strained immediNonstruchrral carbohydrate, % ately through four layers of cheesecloth, the pH 24.4 Enzyme amlysis 38.2 31.3 was determined, and a 200-ml aliquot was 25.4 calculated9 39.2 32.2 Fat," % 9.3 9.7 11.1 acidified and frozen. An additional 200 ml were composited in a vessel containing 4 ml of 'Sodium bicarbonate included at 1.5% of diet DM. 21ncluded a timothy, orchardgrass, and fescue mix; saturated mercuric chloride for microbial harvesting. Bacteria were harvested on d 17 after contained 9.3% protein and 64% NDF. 'Church and Dwight Co., Inc.: 82.5% fat (minimum). the 24-h collection according to the methods described by Smith and McAllan (31). Protozoa 4S, 22%; K, 18%; Mg, 11%. Included to meet a 1 0 1 were harvested from 1 L of strained rumen N:S ratio for each diet. fluid collected 3 h postfeeding. Prior to harvestbcluded to meet or exceed NRC requirements (21) and to maintain a 2:l C z P ratio for each diet. ing, protozoa were prepared and counted (1). The remaining strained fluid was transferred to % l u e s calculated from data of NRC (21). three 500-ml separatory funnels, each contain7Values calculated from data of Pox et al. (6). ing 150 ml of saline supplemented with 5 @. 'Values calculated from data of Reston (26). '[lOO - (CP + (NDF - NDF bound protein) + ash + of dextrose. After incubation at 39'C for 3 h, the protozoa were drawn off and washed three fat)]. times by centrifugation using saline, with the ''~mform-metaano1 extraction. final wash in distilled H 2 0 . On d 16 and 17, TABLE 2. Ingx&ents and chemical composition of expdmental diets on a DM basis.
s3
TABLE 3. In vitro prediction equations used to calculate microbial yield. DM Digestion (%; corrected for microbial matter) = 83.7 - (2.788 NSC) + (2.358 DIP) + (.037 NSC2) Microbial efficiency (B of m/kg digested DM) = -13.5 + (.876 NSC) + (2.672 DIP) - (.011 NSC2) - (.055 D S ) where: NSC = Dietary nonstructural carbohydrate level, percentage of DM. DIP = Dietary degradable intake protein level, percentage of DM. MN = Microbial N.
Journal of Dahy Science Vol. 74, No. 3, 1991
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STOKES ET AL.
TABLE 4. ~ntalreand milk production means for diets1
~~~~
DW
~~
Wd
DMI, % of B W
~~
~~
A B C X A B C -
X
Milk yield, kgld
A
B C
x
22.1 22.9 20.8 21.9 3.7 4.2 3.2 3.7 44.7 43.4 24.6 37.5
21.9 22.3 19.1 21.1 4.0 4.1 3.1 3.7 40.2 39.3 23.5 34.3
16.8 18.3 19.7 18.3 2.7 3.3 3.2 3.1 30.9 34.4 26.0 30.4
NS
.5
NS
.I
NS
1.3
'Diet 1 was formdated to contai0, as percenta%es of DM, 38% nonsln~chnalcarbohydrate (NSC) and 13.7% degradable intake protein PIP);diet 2, 31% NSC and 11.8% DP, diet 3, 24% NSC and 9% DIP. 2 o r t h o g o ~contrasts, w e NS (P > .IO).
cows were bled via jugular venipuncture 3 and 12 h postfeeding. Blood was centrifuged at 2000 x g for 20 min at 4'C. Serum was decanted and frozen until analysis. Cows were milked twice daily with weights recorded at each milking. The mean of the milk yields for d 10 to 17 were used to compare effects of diets.
microbial) and ammonia N in liquid samples (duodenal and m e n ) were determined by AOAC methods (3). Fat in dried diet, ort, and duodenal samples was extracted with chloroform:methanol (2: 1) using automated equipment (Tecator Inc., Hemdon, VA). In addition, Yb-labeled diets and duodenal samples were analyzed for Yb concentration by atomic absorption spectrophotometry following the proc e d w of Hart and Polan (9). Ytterbium conSample Analyses centration in the duodenal samples was used to Diets and orts were dried at 50'C and calculate DM flow to the duodenum and rumiground through a l-mm screen for analyses. nal digestion. Diaminopimelic acid P A P A ) Liquid duodenal samples were homogenized in and RNA concentrations in dried duodenal and two successive wet grinds using a Telanar SD- microbial samples were determined for calcula45 disperser ( T e h a r Co., Cincinnati, OH)first tions of microbial matter by methods of Webfor 8 min using a G450 generator, followed by ster (36) and Zinn and Owens (39), respectivea second grinding for 4 min with a G454 ly. Liquid m e n samples were analyzed for generator. Subsamples were lyophilized and VFA by gas chromatography (34). Serum samground through a 1-mm screen. Diets, orts, and ples were analyzed for urea N concentrations dried duodenal samples were analyzed for NDF photometrically using a S m i W e Diagnostic and ADF by methods of Goering and Van kit (Smith-Kline-Beclanan, Philadelphia, PA) Soest (8) with modification by Robertson and in which diacetylmonoxine was the colorimetVan Soest (27). The NSC content of each diet ric reagent. was determined by both enzyme analysis (30), modified to use ferricyanide as a colorimetric Statistical Analysis agent, and by difference calculations [lo0 Data were analyzed by ANOVA for a 3 x 3 (CP + (NDF - NDF bound protein) + ash + Latin square design using the general linear ether extract)]. Both methods gave similar models procedure of the SAS (32). Model was values; for the rest of this paper, NSC intakes blocked on cows and stage of lactation. Due to and digestibilities are calculated from values ration design, orthogonal contrasts were used to derived by enzyme determination. Total N and test for h e a r and quadratic responses to dOM in dried samples (diet, on, duodenal, and nal availability of carbohydrate and protein. Journal of Dairy Science Vol. 74, No. 3, 1991
875
RUMEN DIGESTION OF PROTEINS AND C A R B O H Y D ~ T E S
TABLE 5. Intakes* and digestibilities of OM, total carbohydrate, and carbohydrate fractions in response to diet2 Diet
SEM
1
2
3
contrast3
19.5
19.5
16.5
NS
"5
13.0 66.6
12.7 65.1
9.2 55.7
NS L
.8 1.4
14.3
14.3
10.5
NS
1.o
9.2 64.1
9.6 67.1
5.8 55.8
NS
NS
.8 4.5
8.8
7.3
4.6
L
.3
6.2 70.6
5.5 74.2
2.2 46.6
L NS
.4 5.9
55
6.9
6.6
NS
.5
2.9 54.0
3.9 565
3.6 55.5
NS
.4
NS
1.5
3.4
3.6
4.2
NS
.4
1.9 45.8
2.0 48.7
2.0 49.9
NS
-1
NS
3.0
OM
Intake. Wd Digested in the rumen4 kgld % OM Intake Total carbohydrate' Intake, kg/d Digested m the m e n kdd % CHO Intake Nonstructural carbohydrate Intake, kg/d Digested in the m e n kdd % NSC Intake NDF Intake, kg/d Digested in the rumen kgld Sb NDF Intake ADP Intake, kg/d Digested in the rumen kgld % ADP Intake
'Intakes adjusted for orts composition. 2Diet 1 was formulated to contain, as percentages of DM, 38% nonstNctural carbohydrate (NSC) and 13.7% degradable htake protein (DIP); diet 2, 31% NSC and 11.8% DP, diet 3, 24% NSC and 9% DIP. 3orthogonal contrasts where L = hear (P < .a) a ~ dNS (P > .IO). 4C0mtcd for microbial OM; calcnlated using RNA analysis of microbial matter. 5 ~ o t a CHO l = struchrnil + Nsc.
with an increase in milk production as did cows A and B, it should be noted that cow C produced similar milk yields throughout the last Intakes, Milk Production, two periods (diets 2 and 1, respectively) in and Dlgestlbllltles spite of the loss of a quarter to mastitis. Dry matter intakes and milk production maAverage intakes and digestibilities of OM ble 4) were not different among diets (P > .lo). and carbohydrate (CHO) fractions are preMilk production was greater with diet 1 > 2 > sented in Table 5. Intakes of OM were similar 3. Lack of statistical differences in intake and for all diets, but OM digested in the rumen milk yield appear due to cow C maintaining decreased as NSC and DIP decreased (P c .05). essentially constant consumption and milk pro- Diets were formulated to provide approxiduction irrespective of diet. Cow C received mately 65% of DM as total CHO, with the difdiets in the order of 3, 2, and 1; on d 12 of ference among diets 1,2, and 3 being in N D F period 2 (diet 2), she was diagnosed with acute NSC ratios, which were 4258, 51:49, and 62: mastitis, which resulted in the loss of a quarter. 36, respectively. Total CHO intake and ruminal To allow for this, period 2 for cow C was digestibility were not different among diets, extended until feed intake returned to normal but, due to diet composition, intake of NSC and was maintained (an additional week). Al- differed (P < .05) among diets. Nonstructural though cow C did not respond to diets l and 2 carbohydrate digestion (percentage of NSC inRESULTS AND DISCUSSION
Journal of Dairy Science Vol. 74, No. 3, 1991
876
STOKES ET AL.
TABLE 6. Ruminal VFA levels, ruminal pH, ruminal NH3 N, and serum
u r ~ aN
as affected by diet'
~~
Diet VFA, mM3
1
2
3
contras?
SEM
142.6
121.4
99.0
L
9.4
65.9 19.5 10.6 1.43 1.39
NS NS NS NS NS NS NS NS
.9
VFA, moVlOO mol
Acetate (A) Propionate (p) Butyrate Isobutyrate Isovalerate Valerate A:P PH3 NH3 N? mg/d senun urea N," mg/dl
56.7 26.9 12.0 1-46 1.35 1.59 2.40 5.9 21.2 19.8
59.9 22.8 12.7 1.31 1.45 1.57 2.66 6.3 15.0 23.4
1.24
3.39 6.6 8.0 23.7
L
Q
.8
.2 .6 .1
.2 .3 .2 1.6 .8
Diel 1 was formulated to contain, as percentages of DM, 38%nonStnrcIuralcarbohydrate (NSC) and 13.7%degradable intake protein PIP);diet 2, 31% NSC and 11.8% DIP; diet 3, 24% NSC and 9% DIP. 2onhogonal contrasts, where L and Q = linear and quadratic (P < .05), respectively, L = linear (P < .lo) and NS (P> .lo). 3Average of five sample times over 1 2 4 period. 4Average of four sample tima over 48-h period.
take) decreased with diet NSC level. Although diets 1 and 2 appeared to be similar, diet 2 did not deviate statistically from a linear line. Of the total CHO digested in the rumen, the NSC fraction accounted for 68% with diet 1, 59% with diet 2, and only 38% with diet 3. Although the major source of starch was similar in all diets (86, 70, and 75% of NSC from ground corn plus corn silage for diets 1, 2, and 3, respectively), diets 1 and 2 averaged 55% higher NSC digestibility compared with diet 3. Others have reported decreased starch digestion in lactation rations with low DIP (11). Ruminal digestion of NSC with diets 1 and 2 was higher than reported in the literature with diets containing >3Wo of DM as NSC and in which corn was the major source (19). Intakes of the fiber fractions (NDF and A D n were lower for diet 1 than for diets 2 and 3, but differences were NS (P > .lo). Digestibility coefficients for NDF and ADF were similar across all diets, averaging 55 and 48%, respectively. Of the total CHO digested in the rumen, the NDF fraction accounted for 32% with diet 1,41% with diet 2, and 62% with diet 3. It appears that when the ruminally digested carbohydrate fraction shifted from >50% NSC to >50% NDF, intake and OM digestion of the J o d of Dairy Science Vol. 74, No. 3, 1991
diets were reduced. The maintenance of fiber digestion (as a percentage of NDF intake) with diets high in NSC agrees with other reports (10, 11) but is in conflict with the results of McCarthy et al. (19), who noted a depression in NDF digestion as ruminal availability of starch inCreaSed. Volatlle Fatty Acld Productlon, Rumlnal Condltlons, Serum Urea Nltrogen, and Mlcroblal Composltlon
Concentrations of total ruminal VFA decreased (P < .lo) as ruminal availability of carbohydrate and protein decreased flable 6). Volatile fatty acid concentration with diet 1 was similar to literature values reported for diets containing starch levels greater than 30% of the ration and supplemented with ruminally degradable protein sources (19). The trend for change in individual molar proportions of VFA (Table 6) reflects the CHO composition of the diets. These differences, however, were NS and indicate that the changes in NDF:NSC ratios in this study did not markedly alter VFA ratios. The high concentration of total VFA in diet 1 is consistent with the low ruminal pH (Table 6) observed with this diet. Ruminal pH below 6 may have a detrimental effect on microbial
877
RUMEN DIGESTION OF PROTEINS AND CARBOHYDRATES
\
---_DIET
\
1
3
t
5.6 0
1
I
I
2
4
6
1
I
I
8 1 0 1 2 HOURS POST FEED
Figure 1. Ruminal pH fluctuations as affected by type of dietary carbohydrate and protein. Diet 1 was formulated to contain, as percentages of DM, 38%nonstruchnal carbohydrate (NSC) and 13.7%degradable intake protein (DE'); diet 2,31% NSC and 11.8%D P , and diet 3,2446 NSC and 9% DIP.
0
L---
0
3 6 9 HOURS POST FEED
12
Figure 2. Ruminal ammonia N concentrations as influenced by type of dietary carbohydrate and protein. Diet 1 was formulated to contain, as percentages of DM, 38% nonstructural carbohydrate (NSC) and 13.7% degradable intake protein (DIP); diet 2.31% NSC and 11.8%DIP, and diet 3, 24% NSC and 9% DIP.
suggesting that factors other than ruminal ammonia level affect blood urea N. reductions are cyclic and of short duration, the Microbial composition is presented in Table depression may be moderate (12). Ruminal pH 7. Bacterial content of N and ash were similar with diet 1 reached a low of 5.7 at 6 h after between diets 1 and 2 but were lower (P< .05) feeding but returned to >6 after t h i s time. and higher (P < .05), respectively, with diet 3. whereas ruminal pH with diets 2 and 3 re- Average bacterial compositions (percentage of mained between 6.2 and 6.8 throughout the day N and of ash) were similar to those reported in (Figure 1). Although the low pH observed with the literature (20), but the decrease in N and diet 1 appeared biologically significant, fiber increase in ash with diet 3 is in contrast to digestion (percentage of MDF intake) was not observations that diet type (forage versus coninhibited. centrate) did not alter microbial composition (7, Ammonia N concentrations (Table 6, Figure 24). Protozoal content of N and ash were not 2) decreased (P< .05) with increasing inclusion affected by diet, and average values were someof protein sources more resistant to ruminal what lower than literature reports (24). degradation. Diet 3 consistently resulted in the Microbial marker concentrations (RNA and lowest N H 3 N levels but remained above 5 mg/ D M A ) were similar across all diets. Bacterial dl even though this diet was formulated for and protozoal concentrations of RNA (11.21 degradable and soluble protein levels of 9.0 and and 7.53% of total N, respectively) were higher 2.5% of dietary DM, respectively. Serum urea than those reported by Arambel et al. (2) and N concentration was significantly lower (P < Ling and Buttery (17), whereas bacteria RNA .01) with diet 1 compared with diets 2 and 3, values were lower than those of Craig et al. (5). growth and cellulolytic activity; however, when
Journal of Dairy Science Vol. 74, No. 3, 1991
878
STOKES ET AL.
TABLE 7. Microbial composition and protozoal numbm as affected by diet.' ~~
I
Diet 2
3
contrast2
8.87 12.53 11.00 .350
8.87 11.74 11.13 .395
8.01 16.30 11.54 .390
Q
.12 .37
NS NS
.m .01
6.44 4.14 7.89
555 4.42 8.79
4.78 4.59 6.75
NS NS
.98 .23
NS
8.1
7.4
3.2
NS
Bacteria
%N %A& RNA N, % BN3 D N A N,4 % BN
Q
SEM
Protozoa
% N % Ash
RNA N, % PN' Protozoa count per ml nunen fluid (X ~
16, ~
~
~~
.w
15
~
'Diet I was formulated to contain, as percentages of DM, 38% nonstructural carbohydrate (NSC) and 13.7% degradable intake protein (DIP); diet 2, 31% NSC and 11.8% D E diet 3, 24% NSC and 9% DIP. 'Orthogonal contrasts, where Q = quadratic ( P < .05) and NS (P > .lo). 3Bacterial N. 4Diaminopimelic acid N. 5pr~t0zoalN.
Diaminopimelic acid concentrations of bacteria (.378% of total bacterial IV) were lower than those of Oluboboken et al. (24). Protozoal numbers (Table 7) were greater, but not significantly greater, with diets 1 and 2 compared with diet 3. The increase in protozoal population with diets 1 and 2 reflects the increase in entodiniomorphs noted with these diets (7.4 x 1 6 , 7.3 x 1 6 for diets 1 and 2, respectively) compared with diet 3 (3.1 x 16). Holotrich count remained unaffected by diet (average, 1.2 x 16). Nitrogen Metabo1ism
Nitrogen intake and degradation results are listed in Table 8. Although nonsignificant, intake of N with diets 1 and 2 averaged 15.5% greater than with diet 3 (P = .23). Due to intake and ruminal degradation differences, DIP levels were 12.5, 12.9, and 6.3% of DM, which were different from the original estimates made using NRC (21) values (13.7, 11.8, and 9.0%, respectively). Ruminal protein digestion was lower for diet 3 (P < .05) than for other diets and was lower than predicted (33.5 vs. 49.9%). Diets were designed to supply a 3.5 kg total CP flow to the duodenum, and flows of nitroge nous compounds to the duodenum were 3.4, 3.5, and 3.7 kg for diets 1,2, and 3, respective Journal of Dairy Science Vol. 74, No. 3, 1991
ly. Recovery of N at the duodenum (duodenal N/N intake) averaged 87% for diets 1 and 2, whereas recovery for diet 3 was 106%,reflecting the LJIP contribution to the duodenal protein supply. Nitrogen data in Table 8 were not corrected for endogenous contribution. Using an average 3.2 g of endogenous N (EN) per kilogram of duodenal DM flow (13, 28) gave estimates of 40.7, 41.1, and 41.2 s/d of EN flow for diets 1, 2, and 3, respectively. Accounting for these estimates of EN contribution, true dietary protein degradation values averaged 74.5, 74.6, and 41.2% for diets 1, 2, and 3, respectively. Microbial N production (Table 8) was determined using both DAPA and RNA concentrations. A criticism of the RNA technique is the common use of the RNA Ntotal N ratio of bacteria to estimate microbial N in duodenal contents containing both bacteria and protozoa. This is thought to give values underestimating actual microbial production because protozoa RNA N:total N ratios have been shown to be somewhat lower than those of bacteria (17). In this study, both bacteria and protozoal fractions were isolated and analyzed and an 80:20 weighted ratio was used in the RNA calculations (25). Although production of microbial N (RNA method) appeared similar between diets 1 and 2 (averaging 61% greater than diet 3).
879
RUMEN DIGESTION OF PROTEINS AND CARBOHYDRATES
TABLE 8. Intake and digestion of N and flow of nitrogenous compounds to the duodenum. Diet
N Intake? g/d Ruminal N digestiq4 % Intake Flow to the duodenum Total N, g NAN. g Microbial N, g RNA DAPA NA"? g RNA DAPA~ Microbial N g/kg OM truly digested RNA DAPA carbohydrate digested RNA DAPA
1
2
3
contrast2
sEh4
633
642
552
NS
34
69.0
68.0
33.5
Q
3.6
542 519
566 537
587 568
NS NS
30 30
317 367
333 376
202 266
L
10 38
201 15 1
204 161
366 302
L
NS
24.8 26.8
26.4 27.6
22.0 26.9
NS NS
35 5 40.6
35.0 39.7
345 45.7
NS NS
~~
23 8
Q
1.6 2.7 3.1 6.3 ~
~~
'Diet 1 was formulated to contain, as percentages of DM, 38% noostructaral carbohydrate (NSC) and 13.7% degradable intake protein (DIP); diet 2, 31% NSC and 11.8% DIP, diet 3, 24% NSC and 9% DIP. %rhogod contrasts where L and Q = limar and quadratic (P < -05). respectively, and NS (P > .lo). for orts composition. 3c0-ted 4Calculated using RNA to estimate microbial matter. %onammoria, nonmicrobial N. 6Diaminopimelic acid.
diet 2 did not deviate statistically from the linear response line. As a fraction of the total duodenal protein flow, microbial protein production supplied 59, 59, and 34% for diets 1,2, and 3, respectively. Microbial yields (RNA method) in this study were in the range of those repurted by McCarthy et al. (19) but lower than those observed by Herrera-Saldana et al. (10). Variation in microbial protein production may be explained partially by differences in methods of estimation, because Herrera-Saldana et al. (10) used an assumed nucleic acid content of microbial N, whereas McCarthy et al. (19), as in this study, measured microbial RNA. The greatest nonammonia, nonmicrobial N flow to the duodenum occurred with diet 3, which included protein sources having greater resistance to ruminal degradation. Microbial efficiencies (Table 8), expressed as grams of microbial N per kilogram of OM truly digested, did not differ among diets. Efficiencies in this study were lower than the
means of 38 and 32 g of microbial N/kg of OM truly digested observed with other studies (10, 19). It has been suggested that microbial yields should be expressed in terms of CHO rather than OM because CHO is the primary source of energy for microbial growth (22). Although microbial efficiencies expressed in this manner averaged 49% greater than those based on weight of OM digested, no differences due to diet type were noted. In summary, ruminal digestibility of OM, NSC, and CP was increased when NSC and DIP were >24 and 9% of DM, respectively; however, digestion was not further improved by increasing the level of NSC greater than 31% of the ration. Microbial protein synthesis responded similarly and was maximized with the NSC and DIP levels in diet 2 (31 and 12.9%of DM as NSC and DIP, respectively). Ruminal fermentation (as measured by OM digestion and VFA concentration) was enhanced with diets containing CHO and protein sources more Journal of Dairy Science Vol. 74, NO. 3, 1991
880
STOKES ET AL.
available in the rumen (diets 1 and 2 compared with diet 3). Microbial efficiency (expressed in terms of weights of OM truly digested or CHO digested) remained unaffected by diet, reflecting the proportionate reductions in both digestion and microbial protein synthesis with decreasing dietary NSC and DIP concentrations. This study was designed to explore the potential for manipulating the flow of fermentation end products and microbial protein from the rumen. Production of microbial N and levels of VFA in the rumen were enhanced by appropriate levels of NSC and DIP, which reduced the amount of bypass protein and fat needed to meet requirements for production. Diets formulated to contain, as percentages of DM, 31 or 39% NSC and 11.8 or 13.7% DIP supported greater microbial protein synthesis and VFA production than did a diet containing 25% NSC and 9% DIP. REFERENCES 1 Abe, M, and P. Kumeno. 1973. In vitro simulation of rumen fermentation: Apparatus and effects of dilution rate and continual dialysis on fermentation and protozoal populations. J. Anim. Sci. 36941. 2Arambel M. J., E. E. Bartley, S. M.Dennis, G. S. Ma,T. G. Naganja, D. E. Nuzback, D. 0. Riddell, A. D. Dayton, and S. J. Galitzer. 1987. Evaluation of several methods for estimating microbial nitrogen concentration in the rumen. Nu@. Rep. Int. 35%. 3Association of Ofticial Analytical Chemists. 1970. official methods of analysis. 11th ed. AOAC. Washinptq Dc. 4 Casper, D. P.,and D. J. Schingoethe. 1989. Lactational response of dairy cows to diets varying m ruminal solubilities of carbohydrate and crude protein. J. Dairy Sci. 72928. 5 Craig, W. M.,D. R Brown,G. A. Broddck and D. B. Ricker. 1987. Post-primdial changes of fluid- and particle-associated ruminal microorganisms. J. Anim. Sci. 65: 1042. 6 Fox,D. G., C. J. Sniffen, J. D. O'Connor. J. B.Russell. and P. J. Van SO&. 1986. The C ~ m e l lMt Carbohydrate and protein system for evaluating cattle diets. Comell Univ., Ithaca, NY. 7Fwchtenicht, J. E., and G. A. Broderick. 1987. Effect of inoculum prepantion and dietary energy on microbial numbem and rumen protein degradation activity. J. Dairy Sci. 701404. 8 Goeriog, H. K.,and P.J. Van Soest. 1970. Forage fiber analyses (apparatus, reagcnls, procedans, and some applications). Agric. Haedbook NO. 379. ARS-USDA,
Washington,Dc. 9% S. P., ami C. E. Polan. 1984. Simultaneous extraction and determination of ytterbium and cobalt ethylenediamineteeetate complex in feces. J. Dairy Journal of Dairy Science Vol. 74, No. 3, 1991
Sci. 67388. lOHerrera-Saldana, R., R. Gomez-Alarcon, M. Torabi, and J. T. Huber. 1990. Influence of synchronizing protein and starch d-on in the m e n on nutrient utilization and microbial protein synthesis. J. Dairy Sci. 73:142. 11Hemra-Saldaua, R, and J. T. Huber. 1989. Influence of varyiug protein and starch degradations on performamx of lactating cow. J. Dairy Sci. 72:1477. 12Hoover. W. H. 1986. chcmical factors involved in ruminal fiber westion. J. Dairy Sci. 692755. 13 Hvelplund, T., and J. h4adsea 1985. Amino acid passage to the small intestine in dairy cows compared with estimates of microbial protein and undegraded dietary protein from analysis on the feed. Acta Agric. Scand. Suppl. 2521. 14 Komarek, R J. 1981. Intestinal canudation of cattle and sheep with a t-shaped canuula designed for total digcsm collection without externalizing digesta flow.J. Anim. Sci. 53:7%. 15Lewis, D. 1962. The interrelationships of individual proteins and carbohydrates during fermentation in the rumen of the sheep. II. The fermentation of starch in the presence of starch or other substances containing nitrogen. J. Agric. Sci. (Camb.) 58:73. 16Lewis, D., and I. W. McDonald. 1958. 'Ihc interrelationships of individual proteins and carbohydrates during fermentation in the rumen of the sheep. I. The fermentation of casein in the presence of starch or other carbohydrate materials. J. Agric. Sci. (Camb.) 51: 108. 17Ling, J. R, and P. J. Buttery. 1978. The simultaneous use of ribonucleic acid, %, 2,&diaminopimelic acid as markers of microbial N entering the duodenum of sheep. Br. J. NUB. 39164. 18 Macl@ G. K.,D. G.Grieve, I. McMillaq and G. C. Smith. 1984. Effect of varying protein and energy densities in complete rations fed to cows in first lactation. J. Dairy Sci 67:1421. 19McCarthy, R D., Jr., T. H.Klusmeyer, J. L. Vicini, J. H.Clark, and D. R Nelson. 1989. Effects of source of protein and carbohydrate on ruminal fermentation and passage of nutrienfs to the small intestbe of lactaring cows. J. Dairy Sci. n 2 0 0 2 . 20Mary, R J., and A. B. McAuan. 1983. A comparison of the chemical composition of mixed bacteria harvested from the liquid and solid fractions of rumen digesta. Br. J. Nutr. 5Q701. 21Natiod Research Council. 1989. Nutrient requirements of dairy cattle. 6th rev. d.Natl. Acad. Sci., Washington, Dc. 22NoccL. I. E., and J. B. Russell. 1988. Protein and energy as an integrated system. Relationship of ruminal protein and carbohydrate availability to microbial synthesis and milk production. J. Dairy Sci. 71:2070. 23 Oldham, J. D. 1984. Protein-energy interrelationships in dairy cows. J. Dairy Sci. 67:1090. 24Oluboboken, 1. A., W.M. Craig, and W. A. Nipper. 1988. QzanrteriStic~Of protozoal and bactesial fractions from microorganisms associated with ruminal fluid or particles. J. Anim. Sci. 66:2701. 25pilgrim, A. F., F. V. Gray, R. A. Weller, and C. B. Belling. 1970. Synthesis of microbial protein in the sheep's rumen and the proportion of dietary nitrogen
RUMEN DIGESTION OF PROTEINS AND CARBOHYDRATES converted into microbial nitrogen. Br. J. Nutr. 24589. 26 Preston, R L. 1989. Typical composition of feeds for cattle, sheep. Feedstuffs 61(41):19. 27Robertson, J. B., and P. J. Van Soest. 1977. Dietary fiber estimation in concentrate feedstuffs. J. Anim. Sci. 45(Suppl. 2):254. 28 Rohr, K., P. Lebzien, H. Schafft, and E. Schulz. 1986. Prediction of duodenal flow of eon-ammonianitrogen and amino acid nitrogen in dairy cows. Livest. Prod. Sci. 1429. 29 Russell, J. B.,C. J. Sniffen, and P. J. Van Soest. 1983. Effect of carbohydrate limitation on degradation and utilization of casein by mixed rumen bacteria. J. D a q Sci. 66:763. 30Smith, D. 1969. Removing and analyzing total eonstructural carbohydrate from plant tissue. Wisconsin Agric. Exp. Stn. Res. Rep. 41:l. 31 Smith,R.H., and A. B. McAllan. 1974. Some factors influencing the chemical composition of mixed rumen bacteria. Br. J. Nutr. 31:27. 32 SASQ User’s Guide: Statistics. 1982. SAS Inst., Inc., Gary, NC. 33 Stokes, S. R.,W. H. Hoover, T. K. Miller, and R. P. Manski. 1991. Impact of carbohydrate and protein
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level on bacterial metabolism in continuous culture. J. Dairy Sci. 74854. 34 Supelco Bulletin 749E. 1975. Supelco, Inc., Bellefonte, PA. 35 Waltz, D. M.,M. D. Stern, and D. J. J.Ug. 1989. Effect of ruminal protein degradation of blood meal and feather meal on the intestinal amino acid supply to the lactating cow. J. Dairy Sci. 721509. 36 Webster, P. M.,W. H. Hoover, and T. K. Miller. 1990. Determination of 2,64amhopimelic acid in biological materials using high performance liquid chromatography. Anim. Feed Sci. Technol. 30:ll. 37 Windscbitl, P. M.. and M. D. Stem. 1988. Evaluation of calcium l i g ~ ~ ~ ~ d f ~ ~ t e - tsoybean r e a t e dmeal as a s o w e of rumen protected protein for dairy cattle. J. Dairy Sci. 71:3310. 38Zerbi~i,E., C. E. Polan, and J. H. Herbeii 1988. & meal on Effecls of dietary soybean meal and f protein digesta in Holstein cows during early and midlactation. J. Dairy Sci. 71:1178. 39 Zinn, R A.. and F. N. Owens. 1982. A rapid procedure for microbial protein estimation. Page 26 in Protein reqimnents of cattle: symposium. F. N. Owens, ed. Oklahoma Agric. Exp. sta MP-109.
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