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Dehydrated Alfalfa in Dairy Cow Diets1
Dehydrated Alfalfa in Dairy Cow Diets 1 S. G. PRICE, L. D. SATTER, and N. A. JORGENSEN US Dairy Forage Research Center, USDA-ARS and Dairy Science Department University of Wisconsin-Madison 53706 ABSTRACT
for the three diets, control, dehydrated alfalfa, and dehydrated alfalfa with urea.
The objective of Experiment 1 was to determine protein degradation in the rumen and amino acid supply to and absorption of amino acids from the intestine of lactating dairy cows receiving supplements of soybean meal or a combination of dehydrated alfalfa and corn gluten meal. Four lactating Holstein c o w s , fitted with ruminal, duodenal, and ileal cannulae, were used in a switchback experiment. Two diets consisting of 50% corn silage and 50% concentrate were fed. One diet contained soybean meal and the other contained a mixture of dehydrated alfalfa and corn gluten meal. It was estimated that 76% of the dietary protein was degraded in the rumen with the soybean meal diet compared with 62% with the dehydrated alfalfa:gluten meal diet. Flow of total amino acids to the duodenum was 13% higher for the dehydrated alfalfa:gluten meal than for the soybean meal diet. Experiment 2 consisted of two trials. The objective of Trial 1 was to measure rumen fermentation products in lactating dairy cows fed diets where dehydrated alfalfa, with or without urea, replaced 40% of the concentrate. The objective of Trial 2 was to measure milk production. milk composition, and plasma amino acids of dairy cows in early lactation fed the same diets as in TriaI 1. Milk production was 34.7, 33.4, and 32.8 kg/d and milk fat was 3.48, 3.58, and 3.63 %
INTRODUCTION
Received December 11, 1986. Accepted August 31, 1987. 1Mention of a trade name, proprietary product, or specific equipment does not constitute a guarantee or warranty by the US Department of Agriculture and does not imply its approval to the exclusion of other products that may be suitable.
1988 J Dairy Sci 71:727-736
Interest is increasing in formulating dairy diets to supply more undegraded dietary protein. The fraction of protein in dehydrated alfalfa (dehy), corn gluten meal (gluten), and soybean meal (SBM) that escapes degradation in the tureen has been estimated to he 45, 55, and 30%, respectively (24). Undegraded dietary protein is presumably of higher value if it is a good source of limiting amino acids. Schwab (25) observed that lysine and methionine infused into the abomasum of lactating Holstein cows fed corn-based diets accounted for 43% of the total response in milk protein yield obtained from infusates of 10 essential amino acids or sodium caseinate. This suggested lysine and methionine are colimiting amino acids with corn-based diets. Dehydrated alfalfa, rich in Iysine, and gluten, rich in methionine, should be a good combination of protein supplements for meeting amino acid requirements of lactating dairy cows fed corn-based diets. Rock (23) observed protein efficiencies, defined as [(daily gain with test protein) (daily gain with urea)I/daily protein intake above urea control, to be .50 and .55 for gluten and dehy and .20 for SBM. These values were obtained during steer growth trials where total dietary protein was 40% test protein and 60% urea. Corn gluten meal and dehy fed together gave a value of .83, indicating complementary effects on amino acid balance. In addition to being a source of protein, dehy can substitute for grain in the dairy diet. Baldwin et al. (3) observed that replacing 20% of a corn-soybean concentrate with ground alfalfa decreased milk production but significantly increased fat test, resulting in no difference in 4% FCM between the two diets. When dehy replaced 40% of a corn-soybean concentrate (20% of the total diet), milk pro-
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duction and 4% FCM increased (P<.05) in early lactation dairy cows (12). Kirkpatrick et al. (21) observed that when 45% of a barley-based concentrate (22.5% of the total ration) was replaced with dehy, there were no detrimental effects on milk production or milk composition. Substitution of dehy for grain could enhance utilization of NPN in the diet because of the relatively low degradability of dehy protein in the rumen. Combination of a resistant protein source such as dehy with NPN may provide an economical substitute for protein (22). This study consisted of two experiments. The objective of Experiment 1 was to measure the utilization of dietary protein by lactating cows supplemented with SBM or dehy:gluten. The objective of Experiment 2 was to measure milk production, milk composition, plasma amino acid concentration, and tureen fermentation products in lactating cows consuming diets supplemented with SBM or dehy with or without urea.
divided into four equal portions fed at 0400, 1000, 1600, and 2200 h. The amount of time cows spent eating, ruminating, and resting was determined by observation once every 5 min during 1 d (24 h). This was done once during each experimental period. Ytterbium served as an indigestible marker. The Yb-marked feed was prepared by dissolving 256 g of YbCI3:6H20 in 4 L of water, which was sprayed onto 5% of the total ration. The marked feed was mixed and fed with the remaining portion of the meal. The concentration of marker element in the diet was 33 mg/kg DM. Samples of concentrate, corn silage, and orts were obtained daily during the collection period and composited for analyses. Dry matter determinations of concentrate and orts were made after drying at 60°C for 48 h (2). Dry matter content of corn silage was determined by toluene distillation (13). Rumen samples were collected via rumen cannulae at 2, 4, and 6 h postfeeding on each of the last 3 d of each period. The pH was de-
MATERIALS AND METHODS Experiment 1
Four lactating Holstein cows fitted with permanent ruminal cannulae and duodenal and ileal T-type cannulae were used as experimental animals in a switchback design. During Period 1, one pair of cows was fed supplemental SBM and the other pair was fed dehy:gluten. Pairs were reversed in Period 2 and in Period 3 reverted to the same assignment as in Period 1. Each experimental period consisted of 14 d with the first 10 d as an adaptation period and the last 4 d as a sampling period. The duodenal cannulae were located anterior to the common pancreatic-bile duct. The ileal cannulae were located approximately 5 cm from the ileocecal junction. Diets consisted of corn silage and concentrate (1:1 on DM basis). Ingredient and chemical compositions of the diets are given in Table 1. The dehy pellets used in this experiment contained 19.6% CP, 48.7% NDF, and 31.3% ADF (DM basis). Less than 10% of the total N was in the acid detergent insoluble N fraction. Daily rations were offered in amounts yielding less than 5% feed refusals and were Journal of Dairy Science Vol. 71, No. 3, 1988
TABLE 1. Ingredient and chemical compositions of soybean meal and dehydrated alfalfa gluten meal (dehy:gluten) diets (Experiment 1).
Ingredient
Soybean meal
Dehy: gluten
Corn silage Ground yellow corn Soybean meal Dehydrated alfalfa Corn gluten meal Dicalcium phosphate Limestone Monosodium phosphate Trace mineralized salt a Vitamin premix2
% (DM Basis) 52.9 52.2 30.3 14.8 15.6 2513 ... ... 6.8 .2 .1 .4 "" 12 ... .5 .5 .1 .1
Chemical composition CP NDF ADF
14.7 27.2 15.2
14.2 37.5 23.5
1Composition (g/100 g): NaC1 (96 to 98.5); Zn (>.35); Mn (>.20); Fe (>.20): Cu (>.03);I (>.007); and Co (>.005). 2Composition (IU/kg): 2,200,000 vitamins A and D; 220 of vitamin E.
DEHYDRATED ALFALFA termined on fresh samples. Rumen fluid was centrifuged at 1000 x g to remove large particles. The supernatant was centrifuged at 20,000 x g, and 50 ml of that supernatant were preserved with 2 ml of 50% sulfuric acid for later analysis of VFA. The pellet remaining after centrifugation at 20,000 x g was rinsed with physiological saline and centrifuged again at 20,000 x g. The final pellet was rinsed with a small amount of distilled water and freezedried. Preparations of rumen bacteria from each cow on each diet were prepared for diaminopimelic acid analyses. Eighteen samples each of duodenal and ileal digesta were collected over the last 4 d of each period. Sampling was distributed over the 24-h day by having at least one sample from each even numbered hour. The digesta samples (400 ml) were obtained by spot sampling from the cannulae, then individually freeze-dried, and equal amounts from each sample were composited on a dry basis. Fecal grab samples were collected at the same time as the ileal samples, dried at 60°C for 48 h, and composited for analyses. All dried samples were ground through a 1-mm screen in a Wiley mill. Absolute DM was determined by drying samples at 105°C for 24 h. Neutral detergent fiber and ADF were determined by the method of Goering and Van Soest (18). Total N was determined on fresh corn silage, dried grain, orts, and duodenal, ileal, and fecal samples by the Kjeldahl procedure (1) modified by the use of Se as a catalyst and a 4% boric acid solution as an absorbent. Amino acid composition of feedstuffs, orts, rumen bacteria, and digesta samples was determined on 50 mg of dried sample. The samples were hydrolyzed in 6 N HC1 at 105°C for 21 h in sealed tubes under N. Following evaporation and dilution in 50 ml of pH 2.2 citrate buffer, the samples were analyzed on a Beckman 6300 amino acid analyzer (Beckman Instruments, Fullerton, CA). Diaminopimelic acid was used as a bacterial marker, and its concentration in rumen bacteria and duodenal digesta was determined by the method of Czerkawski (11). Volatile fatty acid analyses were performed on a Varian (Palo Alto, CA) Vista 6000 gas chromatograph following preparation of acidified ruminal fluid by the method of Erwin et al. (17). The column was
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packed with GP 10% SP-1200/1% H3PO 4 on 80/100 chromosorb. Ammonia was determined by the catalyzed phenol hypochlorite and ninhydrin colorimetric procedures adapted to the Technicon (Tarrytown, NY) auto analyzer as described by Broderick and Kang (6). The concentration of Yb in the marked meals, digesta samples, and fecal samples was determined with a Beckman direct current plasma spectrometer (10). Results were analyzed statistically by using Statistical Analysis System (28) and its General Linear Model procedure. Results from one animal on the SBM diet deviated more than 2 SD from the treatment means and were not used in the final statistical analysis. Treatments with significant F-vatues were compared using least square difference (LSD). The statistical model used was Yijk --/2 + A i +/3j + 7k(ij) + eijk where A i = cow effect; flj = period effect; Tk(ij) = treatment effect; eij k = error term; i = 1, 2, 3, 4,;j = 1, 2, 3 ; a n d k = 1, 2 (26).
Experiment 2
Trial 1. Trial 1 was an incomplete block design with two 10-d periods. The experimental animals were six Holstein cows fitted with permanent ruminal cannulae. The ingredient and chemical compositions of the diets for Trials 1 and 2 are given in Table 2. The grain mix consisting of corn and mineral and vitamin supplements remained constant across diets, and was partially replaced by dehy in the treatment diets. Ruminal fluid was collected via the rumen cannula at 0, 1, 3, and 5 h postfeeding on 2 consecutive d and then strained through four layers of cheesecloth. Digesta pH was determined on fresh samples. Fifty milliliters of the strained ruminal fluid were preserved with 2 mI of 50% sulfuric acid and frozen for later analyses. Volatile fatty acid and ammonia analyses were carried out as in Experiment 1. Trial 2. Trial 2 was a 3 x 3 Latin square replicated three times with three 28-d periods. The experimental animals were nine high producing cows in earl}- lactation. Feed consumption and milk production were recorded daily. Milk samples were collected on 2 consecutive d during wk 3 and 4. Milk protein and milk fat were analyzed by infrared analysis (2). Jugular blood samples were drawn into hepar-
Journal of Dairy Science Vol. 71, No. 3, 1988
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TABLE 2. Ingredient and chemical compositions of experimental diets (Experiments 2, Trials 1 and 2).
Ingredient
Corn silage Ground yellow corn Dehydrated alfalfa Soybean meal Urea Dicalcium phosphate Limestone Trace-mineralized salt 1 Vitamin premix 2 Chemical analyses CP NDF ADF
Controlsoybean meal
50.1 30.9 1717 " " 13 .4 .5 .1 16.8 31.5 16.8
Dehydrated alfalfa % (DM Basis) 50.0 16.3 20.3 12.8
Dehydrated alfalfa + urea
.2
50.0 22.8 20.5 5.1 .9 .3
.3 .1
" " 13 .1
17.1 37.8 21.9
16.8 37.8 21.5
...
1 Composition (g/lO0 g): NaCI (96 to 98.5); Zn (>.35); Mn (>.20); Fe (>.20): Cu (>.03); I (>.007); and Co (>.005). 2 Composition (IU/kg): 2,200,000 vitamins A and D; 220 of vitamin E.
inized t u b e s at 5 h p o s t f e e d i n g to m e a s u r e p l a s m a a m i n o acids. Blood p l a s m a was p r e p a r e d for a m i n o acid analysis b y m i x i n g it w i t h sulfosalicylic acid (SSA) t o give a final conc e n t r a t i o n o f 3% SSA (wt/vol). T h e s u p e r n a t a n t a f t e r c e n t r i f u g a t i o n was used for analysis. A m i n o acid analysis was d o n e o n a B e c k m a n 6 3 0 0 a m i n o acid a n a l y z e r w i t h S-&-aminoethyll-eystine as an i n t e r n a l s t a n d a r d . Statistical analysis was d o n e using Statistical Analysis S y s t e m (28) a n d its G e n e r a l Linear Model procedure. Treatments with significant F-values were c o m p a r e d using LSD. T h e statistical m o d e l for Trial 1 was Yijkl = / l + A i +/3j + ~'k + (~7)jk + ~il + (6A)il + (6t3)jl + eijkl w h e r e / ~ = overall m e a n ; A i = cow e f f e c t ; ~j = t r e a t m e n t effect; k = p e r i o d e f f e c t ; (/35)jk = t r e a t m e n t (period) effect; 6 = h o u r effect; (6A)il = h o u r x cow effect; (6/3)jl = h o u r x t r e a t m e n t effect; eijkl = split-plot e r r o r ; i = 1 . . 6 ; j = 1,2,3,; k = 1,2; a n d 1 = 1 . . 4 . T h e statistical m o d e l f o r Trial 2 was Yijk = /~ + A i + /3j + 7 k + e~k w h e r e /a = overall m e a n ; A i = cow effect; IJj = t r e a t m e n t ; 7 k - p e r i o d effect; eij k = e r r o r t e r m ; i = 1 . . 9 ; j = 1,2,3; a n d k = 1,2,3 (8, 28). Journal of Dairy Science Vol. 71, No. 3, 1988
RESULTS A N D DISCUSSION Experiment 1
Dry m a t t e r i n t a k e s averaged 16.8 and 16.9 k g / d for t h e t w o t r e a t m e n t s ( T a b l e 3). T h e a p p a r e n t DM digestibilities for t h e t w o diets at t h e d u o d e n u m , ileum, and in t h e t o t a l t r a c t were n o t d i f f e r e n t ( P > . 0 5 ) as e v i d e n c e d b y DM flow m e a s u r e m e n t s . Dry m a t t e r digestibilities in t h e t o t a l t r a c t for t h e SBM a n d d e h y : g l u t e n were 62.7 and 57.5%. Organic m a t t e r i n t a k e s f o l l o w e d t h e same t r e n d s as DM. T o t a l c h e w i n g times, including b o t h r u m i n a t i o n a n d eating, averaged 35.4 m i n / k g feed for SBM a n d 34.7 m i n / k g feed for d e h y : g l u t e n diets. T h i s e x p e r i m e n t was n o t i n t e n d e d to evaluate milk p r o d u c t i o n or milk c o m p o s i t i o n responses. T h e i n t e s t i n a l l y c a n n u t a t e d cows available for this e x p e r i m e n t were in mid to late l a c t a t i o n , and t h e r e f o r e , were less sensitive t o d i e t a r y p r o t e i n supply. T h e b o d y weights a n d milk p r o d u c t i o n and milk c o m p o s i t i o n meas u r e m e n t s (Table 4) were n o t c o n s i d e r e d as critical assessments o f t h e diets b e i n g tested. B o d y w e i g h t changes are d i f f i c u l t t o assess in late l a c t a t i o n b e c a u s e increases c o u l d be f r o m f e t a l g r o w t h or fat d e p o s i t i o n .
DEHYDRATED ALFALFA
73 1
TABLE 3. Daily dry matter intake, flow, and digestion in the gastrointestinal tract of cows fed supplements of soybean meal or dehydrated alfalfa and corn gluten meal (dehy:gluten) (Experiment 1). Treatment Item
Soybean meal
Dehy: gluten
Intake, kg/d Flow to duodenum, kg/d Flow to termi,nal ileum, kg/d Fecal output, kg/d Apparent digestibility in stomach, % Apparent digestibility in small intestine, % Apparent digestibility in total gastrointestinal tract, %
16.8 10.3 8.17 6.21 37.9 50.8 62.7
16.9 11.3
SEM* .2 .3
8.90
.4
7.21 33.6
.2 1.8 2.1 1.1
47.7
57.5
1Means represent six observations obtained with four different cows during three periods.
Values for ruminal m e a s u r e m e n t s averaged over 2, 4, and 6 h p o s t f e e d i n g are given in Table 4. The p H was n o t significantly d i f f e r e n t , a l t h o u g h the d e h y : g l u t e n meal diet had a slightly higher pH. A m m o n i a and t o t a l V F A c o n c e n t r a t i o n s were greater ( P < . 0 5 ) w i t h t h e SBM diet, indicating m o r e extensive ruminal
f e r m e n t a t i o n . The SBM diet resulted in significantly m o r e isovalerate and increased i s o b u t y r a t e , suggesting t h a t m o r e p r o t e i n was d e g r a d e d in the r u m e n w i t h this diet t h a n w i t h t h e d e h y : g l u t e n meal diet. Increased ( P < . 0 5 ) a c e t a t e with t h e d e h y : g l u t e n meal diet yielded an a c e t a t e to p r o p i o n a t e ratio o f 3.63 c o m -
TABLE 4. Body weight, milk yield, chemical composition of milk, and ruminal measurements of cows fed supplements of soybean meal or dehy:corn gluten meal (Experiment 1). Treatment Item
Soybean meal
Dehy: gluten
Animal measurements Body weight, kg Milk yield, kg/d Milk fat, % Milk protein, %
731 a 11.6 3.45 3.43
749 b 10.2 3.33 3.69
Rumen measurements pH Ammonia, mM Total VFA, mM Individual VFA, mol/100 tool Acetate Propionate Isobutyrate Butyrate Isovalerate Valerate Acetate:propionate
SEM 1
,9 .4 .1 .1
6.24 7.11 a 104 a
6.34 4.50 b 99.2 b
.03 .1 1.2
62.6 a 18, 5 1.42 14.3 a 2.12 a 1.27 3.38 a
65.7 b 18.0 1.19 12.5 b 1.44 b 1.22 3.63b
.3 .3 .1 .3 .1 .1 .1
a'bMeans in the same row with different superscripts differ (P<.05). 1Means represent six observations obtained with four different cows during three periods. Journal of Dairy Science Vol. 71, No. 3, 1988
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pared with 3.26 for the SBM diet. Both ratios were within the range associated with a normal milk fat test. Total N intake~ intestinal flow, and digestion in cows fed SBM or dehy:gtuten meal diets are in Table 5. The flow of dietary N for the dehy:gluten diet tended to be higher than with the SBM diet, suggesting that more protein escaped degradation in the rumen with the dehy:gluten diet than the SBM diet. Due to variation in measuring duodenal flows, however, no significant differences were detected. Bacterial N was measured using diaminopimelic acid (DAP) as a marker. The protozoal contribution to rumen microbial protein synthesis was not measured and if significant, the degradation of dietary protein could be underestimated. The justification and limitations of DAP as a marker have been investigated by el-Shazly and Hungate (15), Dufva et al. (14), and Cockburn and Williams (9). Cockburn and Williams (9) found dietary ingredients and changes in the rumen microbial population and environment affect DAP concentration. For this reason, DAP was measured in rumen bacteria isolated during each treatment and period. Dufva et al. (14) observed most feedstuffs to be devoid of DAP. Total amino acid (TAA) intake did not differ between the diets (Table 6). The flow of TAA to the duodenum was 15% greater and absorption of TAA increased 29% (P<.07) for the dehy:gluten diet. Estimates of protein degradation in the rumen were obtained from
the difference between TAA and bacterial amino acid flow to the duodenum divided by TAA intake. Degradation values for the total dietary protein of 76 and 62% were obtained for the SBM and dehy:gluten diets. Soybean meal provided about 55% of total dietary N for the SBM diet, and the dehy:gluten provided about 65% of the protein for the other diet. The reduction of protein degradation in the rumen with the dehy:gluten diet is supported by reduced concentrations of ruminal ammonia with the dehy:gluten diet, greater isovalerate concentration with the SBM diet, and increased flow of dietary N to the duodenum with the dehy:gluten diet. Estimates of protein degradation .for individual feedstuffs were not made and would be difficult to obtain. The flow of lysine to the duodenum was similar across both diets, although lysine intake was less (P<.05) for the dehy:gluten diet (Table 7). Intakes were similar for methionine in both diets, but 50% more methionine flowed to the duodenum with the dehy:gluten diet. No precautions were taken to avoid oxidation of methionine in the hydrolysis procedure. The average loss of methionine from acid hydrolysis reported in the literature is 11 to 15% (19, 27). Losses of this magnitude would account for the differences in methionine content of feedstuffs as we measured them and as they have been reported in the literature. In conclusion, this study suggested that 76% of total dietary protein from the SBM diet was degraded in the rumen compared with 62%
TABLE 5. Daily nitrogen intake, flow and digestion in the gastrointestinal tract of cows fed supplements of soybean meal or dehydrated alfalfa and corn gluten meal (dehy:gluten) (Experiment 1). Treatment Nitrogen
Soybean meal
Dehy: gluten
SEM1
Intake, g/d Flow to duodenum, g/d Bacterial N Dietary N2 Flow to terminal ileum, g/d Disappearance in stomach, g/d Disappearance in small intestine, g/d Apparent digestibility in total gastrointestinal tract, %
386 376 273 103 154 10.2 222 61.6
382 426 256 170 163 -44.3 263 60.9
6.2 15.9 14.3 19.8 8.7 15.6 11.1 1.0
1Means represent six observations obtained with four different cows during three periods. 2Includes endogenous and protozoal nitrogen. Journal of Dairy Science Vol. 71, No. 3, 1988
DEHYDRATED ALFALFA
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TABLE 6. Daily amino acid intake, flow, and digestion in the gastrointestinal tract of cows fed supplements of soybean meal or dehydrated alfalfa corn gluten meal (dehy:gluten) (Experiment 1). Treatment Amino acids I
Soybean meal
Dehy: gluten
Intake, g/d Flow to duodenum, g/d Flow to terminal ileum, g/d Fecal output, g/d Disappearance in stomach, g/d Disappearance in small intestine, g/d Apparent digestibility in total gastrointestinal tract, %
2080 1790 717 616 299 a 1070 c 70.6
2160 2060 676
626 97.4 b 1380 d 70.6
SEM 2 82.4 83.0 29.2 38.4 32.5 61.7 2.0
a'bMeans in the same row with different superscripts differ (P<.05). C'dMeans in the same row with different superscripts differ (P<.07). 1Included arginine, histidine, isoleucine, leucine, tryptophan, methionine, phenylalanine, threonine, valine, aspartic acid, serine, glutamic acid, proline, glycine, alanine, tyrosine. 2 Means represent six observations obtained with four different cows during three periods.
f r o m the d e h y : g l u t e n diet. A l t h o u g h total amino acid intake was similar b e t w e e n the two diets, d u o d e n a l flow of total amino acids, b o t h dietary and microbial, was about 13% higher for the d e h y : g l u t e n diet than for the SBM diet. A p p a r e n t absorption of a m i n o acids from the small intestine was 60 and 67% of the T A A entering the small intestine for the SBM and d e h y : g l u t e n diets. The supply o f lysine and m e t h i o n i n e f r o m the d e h y : g l u t e n diet was equal to, or perhaps slightly greater than, the SBM diet. T h e question of w h e t h e r the dehy: gluten diet w o u l d m e e t the needs of cows with
high protein r e q u i r e m e n t s better t h a n the SBM diet remains unanswered.
Experiment 2
Trial 1. No significant differences in pH were measured b e t w e e n t r e a t m e n t s in Trial 1 (Table 8). A m m o n i a c o n c e n t r a t i o n increased (P<.05) with the dehy plus urea diet. This was e x p e c t e d with the addition of NPN. The dehy diet increased (P<.05) total V F A c o n c e n t r a t i o n of rurnen fluid over that of the control diet (Table 8). Kirkpatrick et al. (21)
TABLE 7. Lysine and methionine intake and flow to the duodenum of cows fed supplements of soybean meal or dehydrated alfalfa gluten meal (dehy:gluten) (Experiment 1). Treatment Item
Soybean meal
Deh7: gluten
SEMI
Lysine Intake, g/d Flow to duodenum, g/d
117.6 a 112.4
80.7 b 116.1
5.3 5.9
Methionine Intake, g/d Flow to duodenum, g/d
29.0 28.6
27.2 42.8
1.7 3.5
a'bMeans in the same row with different superscripts differ (P<.05). 1Means represent six observations obtained with four different cows during three periods. Journal of Dairy Science Vo]. 71, No. 3, 1988
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TABLE 8. Rumen measurements of cows fed diets containing soybean meal, dehydrated alfalfa, or dehydrated alfalfa plus urea (Experiment 2, Trial 1). Treatment
Item pH Total VFA, mM Individual VFA, mol/100 mol Acetate Propionate Butyrate lsobutyrate Isovalerate Valerate Acetate:propionate Ammonia, mM
Soybean meal control
Dehydrated alfalfa
Dehydrated alfalfa plus urea
SEM 1
6.18 101 a
6.02 116 b
6.06 113 ab
.01 1.70
61.9 18.8 13.1 1.95 c 2.60 f 1.74 3.30 6.30 a
62.1 18.9 13.3 1.61 d 2.19g 1.71 3.33 5.34 a
63.6 19.3 12.3 1.30 e 1.88 h 1.60 3.36 8.05 b
.10 .07 .06 .02 .02 .02 .01 .14
a'bMeans in the same row with different superscripts differ (P<.05). c'd'eMeans in the same row with different superscripts differ (P<.09). f'g'hMeans in the same row with different superscripts differ (P<.07). 1Means are for nine cows.
observed a similar t r e n d w h e n 45% o f t h e barley grain was replaced w i t h d e h y . The V F A c o n c e n t r a t i o n increased 5.2% w i t h t h a t treatm e n t c o m p a r e d w i t h t h e diet based o n barley grain. I s o b u t y r a t e and isovalerate c o n c e n t r a t i o n s w e r e higher ( P < . 0 9 , P < . 0 7 ) w i t h t h e c o n t r o l diet t h a n w i t h t h e d e h y and d e h y plus urea diet, suggesting m o r e p r o t e i n was d e g r a d e d
in t h e r u m e n w i t h t h e SBM diet. A c e t a t e t o p r o p i o n a t e ratios for all t h r e e diets w e r e w i t h i n t h e range associated w i t h a n o r m a l fat test. Trial 2. Dry m a t t e r intake o f c o w s in Trial 2 averaged 24 kg/d, equaling 4% o f their b o d y w e i g h t (Table 9). A l t h o u g h d i f f e r e n c e s in b o d y weight change o f cows w e r e n o t significant b e t w e e n diets, cows on t h e d e h y and d e h y plus
TABLE 9. Nutrient intakes, body weight gain, and production responses of cows fed diets containing soybean mean, dehydrated alfalfa, or dehydrated alfalfa plus urea (Experiment 2, Trial 2). Treatment
Item
Soybean meal control
Dehydrated alfalfa
Dehydrated alfalfa plus urea
SEM 1
Dry matter intake (DMI), kg/d Neutral detergent fiber intake, kg/d Acid detergent fiber intake, kg/d Body weight gain, kg/d Milk production, kg/d Milk fat, % 4% FCM, kg/d Milk protein, % 4% FCM/DMI, kg/d
24.2 7.6 a 4.0 a .53 34.7 a 3.48 31.6 3.13 a 1.31
23.8 9.0 b 5.2 b .25 33.4 b 3.58 31.0 3.00 b 1.30
24.0 9.2 b 5.2 b .15 32.8 b 3.63 30.7 2.98 b 1.28
.15 .06 .15 .13 .3 .02 .2 .04 .01
a'bMeans in the same row with different superscripts differ (P<.05). 1Means are for nine cows. Journal of Dairy Science Vol. 71, No. 3, 1988
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DEHYDRATED ALFALFA urea diets gained weight more slowly. Changes in body composition could, of course, be important, but these measurements were not made. Milk production was greater (P<.05) with the control diet than with the dehy and dehy plus urea diets (Table 9). The dehy and dehy plus urea diets increased milk fat percent, resulting in no significant difference in 4% FCM between diets. Milk protein of the delay and dehy plus urea diets was reduced (P<.05). Christensen and Cochran (7) also observed reduced milk protein when either 3 or 6 kg of dehydrated alfalfa replaced the barley-based concentrate that was fed with bromegrass alfalfa hay. The reduction in milk protein with dehy addition could reflect lower dietary energy content (16). Plasma concentrations of TAA tended to be higher and nonessential amino acid lower with the dehy diet than with the control diet. The dehy diet increased (P<.05) the plasma concentration of total branched-chain amino acids, individual branched-chain amino acids, and total essential amino acids at 5 h postfeeding compared with the control and dehy plus urea
diets (Table 10). Plasma concentrations of total branched-chain amino acids were increased by 35 and 5% for the dehy and dehy plus urea diets compared with the control diet. This indicates greater protein absorption from the small intestine with the dehy diet than with the control diet. The concentration of amino acids in jugular blood plasma reflects changes in amino acid absorption from the small intestine (5), more so for essential than nonessential amino acids, as the latter can arise from endogenous synthesis (4). Concentrations of branched-chain amino acids in extrahepatic circulation are particularly reflective of absorption, because the portal supply of the amino acids is metabolized to a lesser extent by the liver (20, 29). In summary of Experiment 2, substitution of dehydrated alfalfa for soybean meal and corn resulted in reduced concentration of branched-chain fatty acids in the rumen. This is consistent with a reduction in amino acid degradation by rumen microbes. Based on concentration of branched-chain amino acids in blood plasma, uptake of amino acids from the intestine was improved when dehy replaced
TABLE 10. Plasma amino acid concentration at 5 h postfeeding. Treatment
Amino acid
Soybean meal control
Dehydrated alfalfa
Dehydrated alfalfa plus urea
SEMi
874ab 361 a 513 a 96.6 a 42.8 a 62.2 a 202 a
939 b 450 b 489 ab 133 b 58.4 b 81.9 b 273 b
832 a 364 a 468 b 101a 44.9 a 67.1 a 213 a
13.1 10.1 5.5 3.2 1.8 2.7 7.8
(mM) Total amino acids2 Essential amino acids 3 Nonessential amino acids4 Valine Isoleucine Leucine Total branched-chain amino acids 5
a'bMeans in the same row with different superscripts differ (P<.05). Means are for nine cows. Included essential and nonessential amino acids as described below. 3Included threonine, valine, methionine, isoleucine, leucine, phenylalanine, tryptophan, Iysine, histidine, arginine. 4 Included aspartic acid, serine, asparagine, glutamic acid, glutamine, proline, glycine, alanine, cystine, tyrosine. Journal of Dairy Science Vol. 71, No. 3, 1988
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s o y b e a n meal and corn. Milk p r o d u c t i o n was sustained surprisingly well w h e n d e h y r e p l a c e d c o n c e n t r a t e in t h e diet, w h i c h e m p h a s i z e s t h e n e e d to have m o r e q u a n t i t a t i v e i n f o r m a t i o n on s u b s t i t u t i o n values o f forages f o r c o n c e n t r a t e s u n d e r differing d i e t a r y c o n d i t i o n s .
ACKNOWLEDGMENTS
A p p r e c i a t i o n is e x t e n d e d t o t h e A m e r i c a n Alfalfa Processors A s s o c i a t i o n for their s u p p o r t o f this research and for s u p p l y i n g t h e d e h y d r a t e d alfalfa. REFERENCES
1 Association of Official Analytical Chemists. 1975. Official methods of analysis. 12th ed. Washington, DC. 2 Association of Official Analytical Chemists. 1980. Official methods of analysis. 13th ed. Washington, DC. 3 Baldwin, R., E. M. Kesler, and G. L. Hargrove. 1983. Replacing twenty percent of concentrate with ground hay in an alfalfa-based diet for cows in early lactation. J. Dairy Sci. 66:1069. 4 Bergen, W. G. 1979. Free amino acids in blood of ruminants - physiological and nutritional regulation. J. Anim. Sci. 49:1577. 5 Bergen, W. G., H. A. Henneman, and W. T. Magee. 1973. Effect of dietary protein level and protein source on plasma and tissue free amino acids in growing sheep. J. Nutr. 103:575. 6 Broderick, G. A., and J. H. Kang. 1980. Automated simultaneous determination of ammonia and total amino acids in ruminal fluid and in vitro media. J. Dairy Sci. 63:64. 7 Christensen, D. A., and M. I. Cochran. 1983. Composition and nutritive value of dehydrated alfalfa for lactating dairy cows. J. Dairy Sci. 66:1282. 8 Cochran, W. G., and G. M. Cox. 1957. Page 127 in Experimental designs. 2nd ed. John Wiley & Sons. New York, NY. 9 Cockburn, J. E., and A. P. Williams. 1984. The simultaneous estimation of the amounts of protozoal, bacterial and dietary nitrogen entering the duodenum of steers. Br. J. Nutr. 51 : 111. 10 Combs, D. K. 1985. An evaluation of markers and techniques used to measure nutrient digestion in ruminants. Ph.D. Thesis, Univ. Wisconsin, Madison. 11 Czerkawski, J. W. 1974. Methods for determining 2-6-diaminopimelic acid and 2-aminoethylphosphonic acid in gut contents. J. Sci. Food Agric. 25:1768.
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12 Danelon, J. C. 1983. Evaluation of partial substitution of dietary components with dehydrated alfalfa in diets of lactating dairy cows. M. S. Thesis, Univ. Wisconsin-Madison. 13 DeWar, W. A., and P. McDonald. 1961. Determination of dry matter in silage by distillation with toluene. J. Sci. Food Agric. 12:790. 14 Dufva, G. S., E. E. Bartley, M. J. Arambel, T. G. Nagaraja, S. M. Dennis, S. J. Galitzer, and A. D. Dayton. 1982. Diaminopimelic acid content of feeds and rumen bacteria and its usefulness as a rumen bacterial marker. J. Dairy Sci. 65:1754. 15 el-Shazly, K., and R. E. Hungate. 1966. Method for measuring diaminopimelic acid in total rumen contents and its application to the estimation of bacterial growth. AppI. Microbiol. 14:27. 16 Emery, R. S. 1978. Feeding for increased milk protein. J. Dairy Sci. 61:825. 17 Erwin, E. S., G. J. Marco, and E. M. Emery. 1961. Volatile fatty acid analyses of blood and rumen fluid by gas chromatography. J. Dairy Sci. 44: 1768. 18 Goering, H. K. and P. J. Van Soest. 1970. Forage fiber analysis. USDA, ARS, Agric. Handbook No. 379. 19 Halloran, H. R. 1985. Poultry amino acid needs should be re-evaluated. Feedstuffs 57:10. 20 Harper, A. E., R. H. Miller, and K. P. Block. 1984. Branched-chain amino acid metabolism. Annu. Rev. Nutr. 4:409. 21 Kirkpatrick, B. K., D. A. Christensen, and M. I. Cochran. 1984. Dehydrated alfalfa as a concentrate substitute in rations of lactating dairy cows. J. Dairy Sci. 67:2315. 22 Klopfenstein, T., R. Britton, and R. Stock. 1982. Nebraska Growth System. Page 310 in Proc. Symp. Protein requirements for cattle. 23 Rock, D. W. 1980. Characterization of alfalfa protein for ruminants. Ph.D. Thesis, Univ. Nebraska, Lincoln. 24 Satter, L. D. 1986. Protein supply from undegraded dietary protein. J. Dairy Sci. 69:2734. 25 Schwab, C. G., L. D. Satter, and A. B. Clay. 1976. Response of lactating dairy cows to abomasal infusion of amino acids. J. Dairy Sci. 59:1254. 26 Snedecor, G. W. and W. G. Cochran. 1967. Statistical methods. Iowa State Univ. Press, Ames. 27 Spindler, M. R., Stadler, and H. Tanner. 1984. Amino acid analysis of feedstuffs: determination of methionine and cystine after oxidation with performic acid and hydrolysis. J. Agric. Food Chem. 32:1366. 28 Statistical Analysis System. 1982. ed. SAS Inst., Inc., Cary, NC. 29 Wolff, J. E., E. N. Bergman, and H. H. Williams. 1972. Net metabolism of plasma amino acids by liver and portal drained viscera of fed sheep. Am. J. Physiol. 223:438.
for the three diets, control, dehydrated alfalfa, and dehydrated alfalfa with urea.
The objective of Experiment 1 was to determine protein degradation in the rumen and amino acid supply to and absorption of amino acids from the intestine of lactating dairy cows receiving supplements of soybean meal or a combination of dehydrated alfalfa and corn gluten meal. Four lactating Holstein c o w s , fitted with ruminal, duodenal, and ileal cannulae, were used in a switchback experiment. Two diets consisting of 50% corn silage and 50% concentrate were fed. One diet contained soybean meal and the other contained a mixture of dehydrated alfalfa and corn gluten meal. It was estimated that 76% of the dietary protein was degraded in the rumen with the soybean meal diet compared with 62% with the dehydrated alfalfa:gluten meal diet. Flow of total amino acids to the duodenum was 13% higher for the dehydrated alfalfa:gluten meal than for the soybean meal diet. Experiment 2 consisted of two trials. The objective of Trial 1 was to measure rumen fermentation products in lactating dairy cows fed diets where dehydrated alfalfa, with or without urea, replaced 40% of the concentrate. The objective of Trial 2 was to measure milk production. milk composition, and plasma amino acids of dairy cows in early lactation fed the same diets as in TriaI 1. Milk production was 34.7, 33.4, and 32.8 kg/d and milk fat was 3.48, 3.58, and 3.63 %
INTRODUCTION
Received December 11, 1986. Accepted August 31, 1987. 1Mention of a trade name, proprietary product, or specific equipment does not constitute a guarantee or warranty by the US Department of Agriculture and does not imply its approval to the exclusion of other products that may be suitable.
1988 J Dairy Sci 71:727-736
Interest is increasing in formulating dairy diets to supply more undegraded dietary protein. The fraction of protein in dehydrated alfalfa (dehy), corn gluten meal (gluten), and soybean meal (SBM) that escapes degradation in the tureen has been estimated to he 45, 55, and 30%, respectively (24). Undegraded dietary protein is presumably of higher value if it is a good source of limiting amino acids. Schwab (25) observed that lysine and methionine infused into the abomasum of lactating Holstein cows fed corn-based diets accounted for 43% of the total response in milk protein yield obtained from infusates of 10 essential amino acids or sodium caseinate. This suggested lysine and methionine are colimiting amino acids with corn-based diets. Dehydrated alfalfa, rich in Iysine, and gluten, rich in methionine, should be a good combination of protein supplements for meeting amino acid requirements of lactating dairy cows fed corn-based diets. Rock (23) observed protein efficiencies, defined as [(daily gain with test protein) (daily gain with urea)I/daily protein intake above urea control, to be .50 and .55 for gluten and dehy and .20 for SBM. These values were obtained during steer growth trials where total dietary protein was 40% test protein and 60% urea. Corn gluten meal and dehy fed together gave a value of .83, indicating complementary effects on amino acid balance. In addition to being a source of protein, dehy can substitute for grain in the dairy diet. Baldwin et al. (3) observed that replacing 20% of a corn-soybean concentrate with ground alfalfa decreased milk production but significantly increased fat test, resulting in no difference in 4% FCM between the two diets. When dehy replaced 40% of a corn-soybean concentrate (20% of the total diet), milk pro-
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duction and 4% FCM increased (P<.05) in early lactation dairy cows (12). Kirkpatrick et al. (21) observed that when 45% of a barley-based concentrate (22.5% of the total ration) was replaced with dehy, there were no detrimental effects on milk production or milk composition. Substitution of dehy for grain could enhance utilization of NPN in the diet because of the relatively low degradability of dehy protein in the rumen. Combination of a resistant protein source such as dehy with NPN may provide an economical substitute for protein (22). This study consisted of two experiments. The objective of Experiment 1 was to measure the utilization of dietary protein by lactating cows supplemented with SBM or dehy:gluten. The objective of Experiment 2 was to measure milk production, milk composition, plasma amino acid concentration, and tureen fermentation products in lactating cows consuming diets supplemented with SBM or dehy with or without urea.
divided into four equal portions fed at 0400, 1000, 1600, and 2200 h. The amount of time cows spent eating, ruminating, and resting was determined by observation once every 5 min during 1 d (24 h). This was done once during each experimental period. Ytterbium served as an indigestible marker. The Yb-marked feed was prepared by dissolving 256 g of YbCI3:6H20 in 4 L of water, which was sprayed onto 5% of the total ration. The marked feed was mixed and fed with the remaining portion of the meal. The concentration of marker element in the diet was 33 mg/kg DM. Samples of concentrate, corn silage, and orts were obtained daily during the collection period and composited for analyses. Dry matter determinations of concentrate and orts were made after drying at 60°C for 48 h (2). Dry matter content of corn silage was determined by toluene distillation (13). Rumen samples were collected via rumen cannulae at 2, 4, and 6 h postfeeding on each of the last 3 d of each period. The pH was de-
MATERIALS AND METHODS Experiment 1
Four lactating Holstein cows fitted with permanent ruminal cannulae and duodenal and ileal T-type cannulae were used as experimental animals in a switchback design. During Period 1, one pair of cows was fed supplemental SBM and the other pair was fed dehy:gluten. Pairs were reversed in Period 2 and in Period 3 reverted to the same assignment as in Period 1. Each experimental period consisted of 14 d with the first 10 d as an adaptation period and the last 4 d as a sampling period. The duodenal cannulae were located anterior to the common pancreatic-bile duct. The ileal cannulae were located approximately 5 cm from the ileocecal junction. Diets consisted of corn silage and concentrate (1:1 on DM basis). Ingredient and chemical compositions of the diets are given in Table 1. The dehy pellets used in this experiment contained 19.6% CP, 48.7% NDF, and 31.3% ADF (DM basis). Less than 10% of the total N was in the acid detergent insoluble N fraction. Daily rations were offered in amounts yielding less than 5% feed refusals and were Journal of Dairy Science Vol. 71, No. 3, 1988
TABLE 1. Ingredient and chemical compositions of soybean meal and dehydrated alfalfa gluten meal (dehy:gluten) diets (Experiment 1).
Ingredient
Soybean meal
Dehy: gluten
Corn silage Ground yellow corn Soybean meal Dehydrated alfalfa Corn gluten meal Dicalcium phosphate Limestone Monosodium phosphate Trace mineralized salt a Vitamin premix2
% (DM Basis) 52.9 52.2 30.3 14.8 15.6 2513 ... ... 6.8 .2 .1 .4 "" 12 ... .5 .5 .1 .1
Chemical composition CP NDF ADF
14.7 27.2 15.2
14.2 37.5 23.5
1Composition (g/100 g): NaC1 (96 to 98.5); Zn (>.35); Mn (>.20); Fe (>.20): Cu (>.03);I (>.007); and Co (>.005). 2Composition (IU/kg): 2,200,000 vitamins A and D; 220 of vitamin E.
DEHYDRATED ALFALFA termined on fresh samples. Rumen fluid was centrifuged at 1000 x g to remove large particles. The supernatant was centrifuged at 20,000 x g, and 50 ml of that supernatant were preserved with 2 ml of 50% sulfuric acid for later analysis of VFA. The pellet remaining after centrifugation at 20,000 x g was rinsed with physiological saline and centrifuged again at 20,000 x g. The final pellet was rinsed with a small amount of distilled water and freezedried. Preparations of rumen bacteria from each cow on each diet were prepared for diaminopimelic acid analyses. Eighteen samples each of duodenal and ileal digesta were collected over the last 4 d of each period. Sampling was distributed over the 24-h day by having at least one sample from each even numbered hour. The digesta samples (400 ml) were obtained by spot sampling from the cannulae, then individually freeze-dried, and equal amounts from each sample were composited on a dry basis. Fecal grab samples were collected at the same time as the ileal samples, dried at 60°C for 48 h, and composited for analyses. All dried samples were ground through a 1-mm screen in a Wiley mill. Absolute DM was determined by drying samples at 105°C for 24 h. Neutral detergent fiber and ADF were determined by the method of Goering and Van Soest (18). Total N was determined on fresh corn silage, dried grain, orts, and duodenal, ileal, and fecal samples by the Kjeldahl procedure (1) modified by the use of Se as a catalyst and a 4% boric acid solution as an absorbent. Amino acid composition of feedstuffs, orts, rumen bacteria, and digesta samples was determined on 50 mg of dried sample. The samples were hydrolyzed in 6 N HC1 at 105°C for 21 h in sealed tubes under N. Following evaporation and dilution in 50 ml of pH 2.2 citrate buffer, the samples were analyzed on a Beckman 6300 amino acid analyzer (Beckman Instruments, Fullerton, CA). Diaminopimelic acid was used as a bacterial marker, and its concentration in rumen bacteria and duodenal digesta was determined by the method of Czerkawski (11). Volatile fatty acid analyses were performed on a Varian (Palo Alto, CA) Vista 6000 gas chromatograph following preparation of acidified ruminal fluid by the method of Erwin et al. (17). The column was
729
packed with GP 10% SP-1200/1% H3PO 4 on 80/100 chromosorb. Ammonia was determined by the catalyzed phenol hypochlorite and ninhydrin colorimetric procedures adapted to the Technicon (Tarrytown, NY) auto analyzer as described by Broderick and Kang (6). The concentration of Yb in the marked meals, digesta samples, and fecal samples was determined with a Beckman direct current plasma spectrometer (10). Results were analyzed statistically by using Statistical Analysis System (28) and its General Linear Model procedure. Results from one animal on the SBM diet deviated more than 2 SD from the treatment means and were not used in the final statistical analysis. Treatments with significant F-vatues were compared using least square difference (LSD). The statistical model used was Yijk --/2 + A i +/3j + 7k(ij) + eijk where A i = cow effect; flj = period effect; Tk(ij) = treatment effect; eij k = error term; i = 1, 2, 3, 4,;j = 1, 2, 3 ; a n d k = 1, 2 (26).
Experiment 2
Trial 1. Trial 1 was an incomplete block design with two 10-d periods. The experimental animals were six Holstein cows fitted with permanent ruminal cannulae. The ingredient and chemical compositions of the diets for Trials 1 and 2 are given in Table 2. The grain mix consisting of corn and mineral and vitamin supplements remained constant across diets, and was partially replaced by dehy in the treatment diets. Ruminal fluid was collected via the rumen cannula at 0, 1, 3, and 5 h postfeeding on 2 consecutive d and then strained through four layers of cheesecloth. Digesta pH was determined on fresh samples. Fifty milliliters of the strained ruminal fluid were preserved with 2 mI of 50% sulfuric acid and frozen for later analyses. Volatile fatty acid and ammonia analyses were carried out as in Experiment 1. Trial 2. Trial 2 was a 3 x 3 Latin square replicated three times with three 28-d periods. The experimental animals were nine high producing cows in earl}- lactation. Feed consumption and milk production were recorded daily. Milk samples were collected on 2 consecutive d during wk 3 and 4. Milk protein and milk fat were analyzed by infrared analysis (2). Jugular blood samples were drawn into hepar-
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TABLE 2. Ingredient and chemical compositions of experimental diets (Experiments 2, Trials 1 and 2).
Ingredient
Corn silage Ground yellow corn Dehydrated alfalfa Soybean meal Urea Dicalcium phosphate Limestone Trace-mineralized salt 1 Vitamin premix 2 Chemical analyses CP NDF ADF
Controlsoybean meal
50.1 30.9 1717 " " 13 .4 .5 .1 16.8 31.5 16.8
Dehydrated alfalfa % (DM Basis) 50.0 16.3 20.3 12.8
Dehydrated alfalfa + urea
.2
50.0 22.8 20.5 5.1 .9 .3
.3 .1
" " 13 .1
17.1 37.8 21.9
16.8 37.8 21.5
...
1 Composition (g/lO0 g): NaCI (96 to 98.5); Zn (>.35); Mn (>.20); Fe (>.20): Cu (>.03); I (>.007); and Co (>.005). 2 Composition (IU/kg): 2,200,000 vitamins A and D; 220 of vitamin E.
inized t u b e s at 5 h p o s t f e e d i n g to m e a s u r e p l a s m a a m i n o acids. Blood p l a s m a was p r e p a r e d for a m i n o acid analysis b y m i x i n g it w i t h sulfosalicylic acid (SSA) t o give a final conc e n t r a t i o n o f 3% SSA (wt/vol). T h e s u p e r n a t a n t a f t e r c e n t r i f u g a t i o n was used for analysis. A m i n o acid analysis was d o n e o n a B e c k m a n 6 3 0 0 a m i n o acid a n a l y z e r w i t h S-&-aminoethyll-eystine as an i n t e r n a l s t a n d a r d . Statistical analysis was d o n e using Statistical Analysis S y s t e m (28) a n d its G e n e r a l Linear Model procedure. Treatments with significant F-values were c o m p a r e d using LSD. T h e statistical m o d e l for Trial 1 was Yijkl = / l + A i +/3j + ~'k + (~7)jk + ~il + (6A)il + (6t3)jl + eijkl w h e r e / ~ = overall m e a n ; A i = cow e f f e c t ; ~j = t r e a t m e n t effect; k = p e r i o d e f f e c t ; (/35)jk = t r e a t m e n t (period) effect; 6 = h o u r effect; (6A)il = h o u r x cow effect; (6/3)jl = h o u r x t r e a t m e n t effect; eijkl = split-plot e r r o r ; i = 1 . . 6 ; j = 1,2,3,; k = 1,2; a n d 1 = 1 . . 4 . T h e statistical m o d e l f o r Trial 2 was Yijk = /~ + A i + /3j + 7 k + e~k w h e r e /a = overall m e a n ; A i = cow effect; IJj = t r e a t m e n t ; 7 k - p e r i o d effect; eij k = e r r o r t e r m ; i = 1 . . 9 ; j = 1,2,3; a n d k = 1,2,3 (8, 28). Journal of Dairy Science Vol. 71, No. 3, 1988
RESULTS A N D DISCUSSION Experiment 1
Dry m a t t e r i n t a k e s averaged 16.8 and 16.9 k g / d for t h e t w o t r e a t m e n t s ( T a b l e 3). T h e a p p a r e n t DM digestibilities for t h e t w o diets at t h e d u o d e n u m , ileum, and in t h e t o t a l t r a c t were n o t d i f f e r e n t ( P > . 0 5 ) as e v i d e n c e d b y DM flow m e a s u r e m e n t s . Dry m a t t e r digestibilities in t h e t o t a l t r a c t for t h e SBM a n d d e h y : g l u t e n were 62.7 and 57.5%. Organic m a t t e r i n t a k e s f o l l o w e d t h e same t r e n d s as DM. T o t a l c h e w i n g times, including b o t h r u m i n a t i o n a n d eating, averaged 35.4 m i n / k g feed for SBM a n d 34.7 m i n / k g feed for d e h y : g l u t e n diets. T h i s e x p e r i m e n t was n o t i n t e n d e d to evaluate milk p r o d u c t i o n or milk c o m p o s i t i o n responses. T h e i n t e s t i n a l l y c a n n u t a t e d cows available for this e x p e r i m e n t were in mid to late l a c t a t i o n , and t h e r e f o r e , were less sensitive t o d i e t a r y p r o t e i n supply. T h e b o d y weights a n d milk p r o d u c t i o n and milk c o m p o s i t i o n meas u r e m e n t s (Table 4) were n o t c o n s i d e r e d as critical assessments o f t h e diets b e i n g tested. B o d y w e i g h t changes are d i f f i c u l t t o assess in late l a c t a t i o n b e c a u s e increases c o u l d be f r o m f e t a l g r o w t h or fat d e p o s i t i o n .
DEHYDRATED ALFALFA
73 1
TABLE 3. Daily dry matter intake, flow, and digestion in the gastrointestinal tract of cows fed supplements of soybean meal or dehydrated alfalfa and corn gluten meal (dehy:gluten) (Experiment 1). Treatment Item
Soybean meal
Dehy: gluten
Intake, kg/d Flow to duodenum, kg/d Flow to termi,nal ileum, kg/d Fecal output, kg/d Apparent digestibility in stomach, % Apparent digestibility in small intestine, % Apparent digestibility in total gastrointestinal tract, %
16.8 10.3 8.17 6.21 37.9 50.8 62.7
16.9 11.3
SEM* .2 .3
8.90
.4
7.21 33.6
.2 1.8 2.1 1.1
47.7
57.5
1Means represent six observations obtained with four different cows during three periods.
Values for ruminal m e a s u r e m e n t s averaged over 2, 4, and 6 h p o s t f e e d i n g are given in Table 4. The p H was n o t significantly d i f f e r e n t , a l t h o u g h the d e h y : g l u t e n meal diet had a slightly higher pH. A m m o n i a and t o t a l V F A c o n c e n t r a t i o n s were greater ( P < . 0 5 ) w i t h t h e SBM diet, indicating m o r e extensive ruminal
f e r m e n t a t i o n . The SBM diet resulted in significantly m o r e isovalerate and increased i s o b u t y r a t e , suggesting t h a t m o r e p r o t e i n was d e g r a d e d in the r u m e n w i t h this diet t h a n w i t h t h e d e h y : g l u t e n meal diet. Increased ( P < . 0 5 ) a c e t a t e with t h e d e h y : g l u t e n meal diet yielded an a c e t a t e to p r o p i o n a t e ratio o f 3.63 c o m -
TABLE 4. Body weight, milk yield, chemical composition of milk, and ruminal measurements of cows fed supplements of soybean meal or dehy:corn gluten meal (Experiment 1). Treatment Item
Soybean meal
Dehy: gluten
Animal measurements Body weight, kg Milk yield, kg/d Milk fat, % Milk protein, %
731 a 11.6 3.45 3.43
749 b 10.2 3.33 3.69
Rumen measurements pH Ammonia, mM Total VFA, mM Individual VFA, mol/100 tool Acetate Propionate Isobutyrate Butyrate Isovalerate Valerate Acetate:propionate
SEM 1
,9 .4 .1 .1
6.24 7.11 a 104 a
6.34 4.50 b 99.2 b
.03 .1 1.2
62.6 a 18, 5 1.42 14.3 a 2.12 a 1.27 3.38 a
65.7 b 18.0 1.19 12.5 b 1.44 b 1.22 3.63b
.3 .3 .1 .3 .1 .1 .1
a'bMeans in the same row with different superscripts differ (P<.05). 1Means represent six observations obtained with four different cows during three periods. Journal of Dairy Science Vol. 71, No. 3, 1988
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PRICE ET AL.
pared with 3.26 for the SBM diet. Both ratios were within the range associated with a normal milk fat test. Total N intake~ intestinal flow, and digestion in cows fed SBM or dehy:gtuten meal diets are in Table 5. The flow of dietary N for the dehy:gluten diet tended to be higher than with the SBM diet, suggesting that more protein escaped degradation in the rumen with the dehy:gluten diet than the SBM diet. Due to variation in measuring duodenal flows, however, no significant differences were detected. Bacterial N was measured using diaminopimelic acid (DAP) as a marker. The protozoal contribution to rumen microbial protein synthesis was not measured and if significant, the degradation of dietary protein could be underestimated. The justification and limitations of DAP as a marker have been investigated by el-Shazly and Hungate (15), Dufva et al. (14), and Cockburn and Williams (9). Cockburn and Williams (9) found dietary ingredients and changes in the rumen microbial population and environment affect DAP concentration. For this reason, DAP was measured in rumen bacteria isolated during each treatment and period. Dufva et al. (14) observed most feedstuffs to be devoid of DAP. Total amino acid (TAA) intake did not differ between the diets (Table 6). The flow of TAA to the duodenum was 15% greater and absorption of TAA increased 29% (P<.07) for the dehy:gluten diet. Estimates of protein degradation in the rumen were obtained from
the difference between TAA and bacterial amino acid flow to the duodenum divided by TAA intake. Degradation values for the total dietary protein of 76 and 62% were obtained for the SBM and dehy:gluten diets. Soybean meal provided about 55% of total dietary N for the SBM diet, and the dehy:gluten provided about 65% of the protein for the other diet. The reduction of protein degradation in the rumen with the dehy:gluten diet is supported by reduced concentrations of ruminal ammonia with the dehy:gluten diet, greater isovalerate concentration with the SBM diet, and increased flow of dietary N to the duodenum with the dehy:gluten diet. Estimates of protein degradation .for individual feedstuffs were not made and would be difficult to obtain. The flow of lysine to the duodenum was similar across both diets, although lysine intake was less (P<.05) for the dehy:gluten diet (Table 7). Intakes were similar for methionine in both diets, but 50% more methionine flowed to the duodenum with the dehy:gluten diet. No precautions were taken to avoid oxidation of methionine in the hydrolysis procedure. The average loss of methionine from acid hydrolysis reported in the literature is 11 to 15% (19, 27). Losses of this magnitude would account for the differences in methionine content of feedstuffs as we measured them and as they have been reported in the literature. In conclusion, this study suggested that 76% of total dietary protein from the SBM diet was degraded in the rumen compared with 62%
TABLE 5. Daily nitrogen intake, flow and digestion in the gastrointestinal tract of cows fed supplements of soybean meal or dehydrated alfalfa and corn gluten meal (dehy:gluten) (Experiment 1). Treatment Nitrogen
Soybean meal
Dehy: gluten
SEM1
Intake, g/d Flow to duodenum, g/d Bacterial N Dietary N2 Flow to terminal ileum, g/d Disappearance in stomach, g/d Disappearance in small intestine, g/d Apparent digestibility in total gastrointestinal tract, %
386 376 273 103 154 10.2 222 61.6
382 426 256 170 163 -44.3 263 60.9
6.2 15.9 14.3 19.8 8.7 15.6 11.1 1.0
1Means represent six observations obtained with four different cows during three periods. 2Includes endogenous and protozoal nitrogen. Journal of Dairy Science Vol. 71, No. 3, 1988
DEHYDRATED ALFALFA
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TABLE 6. Daily amino acid intake, flow, and digestion in the gastrointestinal tract of cows fed supplements of soybean meal or dehydrated alfalfa corn gluten meal (dehy:gluten) (Experiment 1). Treatment Amino acids I
Soybean meal
Dehy: gluten
Intake, g/d Flow to duodenum, g/d Flow to terminal ileum, g/d Fecal output, g/d Disappearance in stomach, g/d Disappearance in small intestine, g/d Apparent digestibility in total gastrointestinal tract, %
2080 1790 717 616 299 a 1070 c 70.6
2160 2060 676
626 97.4 b 1380 d 70.6
SEM 2 82.4 83.0 29.2 38.4 32.5 61.7 2.0
a'bMeans in the same row with different superscripts differ (P<.05). C'dMeans in the same row with different superscripts differ (P<.07). 1Included arginine, histidine, isoleucine, leucine, tryptophan, methionine, phenylalanine, threonine, valine, aspartic acid, serine, glutamic acid, proline, glycine, alanine, tyrosine. 2 Means represent six observations obtained with four different cows during three periods.
f r o m the d e h y : g l u t e n diet. A l t h o u g h total amino acid intake was similar b e t w e e n the two diets, d u o d e n a l flow of total amino acids, b o t h dietary and microbial, was about 13% higher for the d e h y : g l u t e n diet than for the SBM diet. A p p a r e n t absorption of a m i n o acids from the small intestine was 60 and 67% of the T A A entering the small intestine for the SBM and d e h y : g l u t e n diets. The supply o f lysine and m e t h i o n i n e f r o m the d e h y : g l u t e n diet was equal to, or perhaps slightly greater than, the SBM diet. T h e question of w h e t h e r the dehy: gluten diet w o u l d m e e t the needs of cows with
high protein r e q u i r e m e n t s better t h a n the SBM diet remains unanswered.
Experiment 2
Trial 1. No significant differences in pH were measured b e t w e e n t r e a t m e n t s in Trial 1 (Table 8). A m m o n i a c o n c e n t r a t i o n increased (P<.05) with the dehy plus urea diet. This was e x p e c t e d with the addition of NPN. The dehy diet increased (P<.05) total V F A c o n c e n t r a t i o n of rurnen fluid over that of the control diet (Table 8). Kirkpatrick et al. (21)
TABLE 7. Lysine and methionine intake and flow to the duodenum of cows fed supplements of soybean meal or dehydrated alfalfa gluten meal (dehy:gluten) (Experiment 1). Treatment Item
Soybean meal
Deh7: gluten
SEMI
Lysine Intake, g/d Flow to duodenum, g/d
117.6 a 112.4
80.7 b 116.1
5.3 5.9
Methionine Intake, g/d Flow to duodenum, g/d
29.0 28.6
27.2 42.8
1.7 3.5
a'bMeans in the same row with different superscripts differ (P<.05). 1Means represent six observations obtained with four different cows during three periods. Journal of Dairy Science Vo]. 71, No. 3, 1988
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PRICE ET AL.
TABLE 8. Rumen measurements of cows fed diets containing soybean meal, dehydrated alfalfa, or dehydrated alfalfa plus urea (Experiment 2, Trial 1). Treatment
Item pH Total VFA, mM Individual VFA, mol/100 mol Acetate Propionate Butyrate lsobutyrate Isovalerate Valerate Acetate:propionate Ammonia, mM
Soybean meal control
Dehydrated alfalfa
Dehydrated alfalfa plus urea
SEM 1
6.18 101 a
6.02 116 b
6.06 113 ab
.01 1.70
61.9 18.8 13.1 1.95 c 2.60 f 1.74 3.30 6.30 a
62.1 18.9 13.3 1.61 d 2.19g 1.71 3.33 5.34 a
63.6 19.3 12.3 1.30 e 1.88 h 1.60 3.36 8.05 b
.10 .07 .06 .02 .02 .02 .01 .14
a'bMeans in the same row with different superscripts differ (P<.05). c'd'eMeans in the same row with different superscripts differ (P<.09). f'g'hMeans in the same row with different superscripts differ (P<.07). 1Means are for nine cows.
observed a similar t r e n d w h e n 45% o f t h e barley grain was replaced w i t h d e h y . The V F A c o n c e n t r a t i o n increased 5.2% w i t h t h a t treatm e n t c o m p a r e d w i t h t h e diet based o n barley grain. I s o b u t y r a t e and isovalerate c o n c e n t r a t i o n s w e r e higher ( P < . 0 9 , P < . 0 7 ) w i t h t h e c o n t r o l diet t h a n w i t h t h e d e h y and d e h y plus urea diet, suggesting m o r e p r o t e i n was d e g r a d e d
in t h e r u m e n w i t h t h e SBM diet. A c e t a t e t o p r o p i o n a t e ratios for all t h r e e diets w e r e w i t h i n t h e range associated w i t h a n o r m a l fat test. Trial 2. Dry m a t t e r intake o f c o w s in Trial 2 averaged 24 kg/d, equaling 4% o f their b o d y w e i g h t (Table 9). A l t h o u g h d i f f e r e n c e s in b o d y weight change o f cows w e r e n o t significant b e t w e e n diets, cows on t h e d e h y and d e h y plus
TABLE 9. Nutrient intakes, body weight gain, and production responses of cows fed diets containing soybean mean, dehydrated alfalfa, or dehydrated alfalfa plus urea (Experiment 2, Trial 2). Treatment
Item
Soybean meal control
Dehydrated alfalfa
Dehydrated alfalfa plus urea
SEM 1
Dry matter intake (DMI), kg/d Neutral detergent fiber intake, kg/d Acid detergent fiber intake, kg/d Body weight gain, kg/d Milk production, kg/d Milk fat, % 4% FCM, kg/d Milk protein, % 4% FCM/DMI, kg/d
24.2 7.6 a 4.0 a .53 34.7 a 3.48 31.6 3.13 a 1.31
23.8 9.0 b 5.2 b .25 33.4 b 3.58 31.0 3.00 b 1.30
24.0 9.2 b 5.2 b .15 32.8 b 3.63 30.7 2.98 b 1.28
.15 .06 .15 .13 .3 .02 .2 .04 .01
a'bMeans in the same row with different superscripts differ (P<.05). 1Means are for nine cows. Journal of Dairy Science Vol. 71, No. 3, 1988
735
DEHYDRATED ALFALFA urea diets gained weight more slowly. Changes in body composition could, of course, be important, but these measurements were not made. Milk production was greater (P<.05) with the control diet than with the dehy and dehy plus urea diets (Table 9). The dehy and dehy plus urea diets increased milk fat percent, resulting in no significant difference in 4% FCM between diets. Milk protein of the delay and dehy plus urea diets was reduced (P<.05). Christensen and Cochran (7) also observed reduced milk protein when either 3 or 6 kg of dehydrated alfalfa replaced the barley-based concentrate that was fed with bromegrass alfalfa hay. The reduction in milk protein with dehy addition could reflect lower dietary energy content (16). Plasma concentrations of TAA tended to be higher and nonessential amino acid lower with the dehy diet than with the control diet. The dehy diet increased (P<.05) the plasma concentration of total branched-chain amino acids, individual branched-chain amino acids, and total essential amino acids at 5 h postfeeding compared with the control and dehy plus urea
diets (Table 10). Plasma concentrations of total branched-chain amino acids were increased by 35 and 5% for the dehy and dehy plus urea diets compared with the control diet. This indicates greater protein absorption from the small intestine with the dehy diet than with the control diet. The concentration of amino acids in jugular blood plasma reflects changes in amino acid absorption from the small intestine (5), more so for essential than nonessential amino acids, as the latter can arise from endogenous synthesis (4). Concentrations of branched-chain amino acids in extrahepatic circulation are particularly reflective of absorption, because the portal supply of the amino acids is metabolized to a lesser extent by the liver (20, 29). In summary of Experiment 2, substitution of dehydrated alfalfa for soybean meal and corn resulted in reduced concentration of branched-chain fatty acids in the rumen. This is consistent with a reduction in amino acid degradation by rumen microbes. Based on concentration of branched-chain amino acids in blood plasma, uptake of amino acids from the intestine was improved when dehy replaced
TABLE 10. Plasma amino acid concentration at 5 h postfeeding. Treatment
Amino acid
Soybean meal control
Dehydrated alfalfa
Dehydrated alfalfa plus urea
SEMi
874ab 361 a 513 a 96.6 a 42.8 a 62.2 a 202 a
939 b 450 b 489 ab 133 b 58.4 b 81.9 b 273 b
832 a 364 a 468 b 101a 44.9 a 67.1 a 213 a
13.1 10.1 5.5 3.2 1.8 2.7 7.8
(mM) Total amino acids2 Essential amino acids 3 Nonessential amino acids4 Valine Isoleucine Leucine Total branched-chain amino acids 5
a'bMeans in the same row with different superscripts differ (P<.05). Means are for nine cows. Included essential and nonessential amino acids as described below. 3Included threonine, valine, methionine, isoleucine, leucine, phenylalanine, tryptophan, Iysine, histidine, arginine. 4 Included aspartic acid, serine, asparagine, glutamic acid, glutamine, proline, glycine, alanine, cystine, tyrosine. Journal of Dairy Science Vol. 71, No. 3, 1988
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PRICE ET AL.
s o y b e a n meal and corn. Milk p r o d u c t i o n was sustained surprisingly well w h e n d e h y r e p l a c e d c o n c e n t r a t e in t h e diet, w h i c h e m p h a s i z e s t h e n e e d to have m o r e q u a n t i t a t i v e i n f o r m a t i o n on s u b s t i t u t i o n values o f forages f o r c o n c e n t r a t e s u n d e r differing d i e t a r y c o n d i t i o n s .
ACKNOWLEDGMENTS
A p p r e c i a t i o n is e x t e n d e d t o t h e A m e r i c a n Alfalfa Processors A s s o c i a t i o n for their s u p p o r t o f this research and for s u p p l y i n g t h e d e h y d r a t e d alfalfa. REFERENCES
1 Association of Official Analytical Chemists. 1975. Official methods of analysis. 12th ed. Washington, DC. 2 Association of Official Analytical Chemists. 1980. Official methods of analysis. 13th ed. Washington, DC. 3 Baldwin, R., E. M. Kesler, and G. L. Hargrove. 1983. Replacing twenty percent of concentrate with ground hay in an alfalfa-based diet for cows in early lactation. J. Dairy Sci. 66:1069. 4 Bergen, W. G. 1979. Free amino acids in blood of ruminants - physiological and nutritional regulation. J. Anim. Sci. 49:1577. 5 Bergen, W. G., H. A. Henneman, and W. T. Magee. 1973. Effect of dietary protein level and protein source on plasma and tissue free amino acids in growing sheep. J. Nutr. 103:575. 6 Broderick, G. A., and J. H. Kang. 1980. Automated simultaneous determination of ammonia and total amino acids in ruminal fluid and in vitro media. J. Dairy Sci. 63:64. 7 Christensen, D. A., and M. I. Cochran. 1983. Composition and nutritive value of dehydrated alfalfa for lactating dairy cows. J. Dairy Sci. 66:1282. 8 Cochran, W. G., and G. M. Cox. 1957. Page 127 in Experimental designs. 2nd ed. John Wiley & Sons. New York, NY. 9 Cockburn, J. E., and A. P. Williams. 1984. The simultaneous estimation of the amounts of protozoal, bacterial and dietary nitrogen entering the duodenum of steers. Br. J. Nutr. 51 : 111. 10 Combs, D. K. 1985. An evaluation of markers and techniques used to measure nutrient digestion in ruminants. Ph.D. Thesis, Univ. Wisconsin, Madison. 11 Czerkawski, J. W. 1974. Methods for determining 2-6-diaminopimelic acid and 2-aminoethylphosphonic acid in gut contents. J. Sci. Food Agric. 25:1768.
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12 Danelon, J. C. 1983. Evaluation of partial substitution of dietary components with dehydrated alfalfa in diets of lactating dairy cows. M. S. Thesis, Univ. Wisconsin-Madison. 13 DeWar, W. A., and P. McDonald. 1961. Determination of dry matter in silage by distillation with toluene. J. Sci. Food Agric. 12:790. 14 Dufva, G. S., E. E. Bartley, M. J. Arambel, T. G. Nagaraja, S. M. Dennis, S. J. Galitzer, and A. D. Dayton. 1982. Diaminopimelic acid content of feeds and rumen bacteria and its usefulness as a rumen bacterial marker. J. Dairy Sci. 65:1754. 15 el-Shazly, K., and R. E. Hungate. 1966. Method for measuring diaminopimelic acid in total rumen contents and its application to the estimation of bacterial growth. AppI. Microbiol. 14:27. 16 Emery, R. S. 1978. Feeding for increased milk protein. J. Dairy Sci. 61:825. 17 Erwin, E. S., G. J. Marco, and E. M. Emery. 1961. Volatile fatty acid analyses of blood and rumen fluid by gas chromatography. J. Dairy Sci. 44: 1768. 18 Goering, H. K. and P. J. Van Soest. 1970. Forage fiber analysis. USDA, ARS, Agric. Handbook No. 379. 19 Halloran, H. R. 1985. Poultry amino acid needs should be re-evaluated. Feedstuffs 57:10. 20 Harper, A. E., R. H. Miller, and K. P. Block. 1984. Branched-chain amino acid metabolism. Annu. Rev. Nutr. 4:409. 21 Kirkpatrick, B. K., D. A. Christensen, and M. I. Cochran. 1984. Dehydrated alfalfa as a concentrate substitute in rations of lactating dairy cows. J. Dairy Sci. 67:2315. 22 Klopfenstein, T., R. Britton, and R. Stock. 1982. Nebraska Growth System. Page 310 in Proc. Symp. Protein requirements for cattle. 23 Rock, D. W. 1980. Characterization of alfalfa protein for ruminants. Ph.D. Thesis, Univ. Nebraska, Lincoln. 24 Satter, L. D. 1986. Protein supply from undegraded dietary protein. J. Dairy Sci. 69:2734. 25 Schwab, C. G., L. D. Satter, and A. B. Clay. 1976. Response of lactating dairy cows to abomasal infusion of amino acids. J. Dairy Sci. 59:1254. 26 Snedecor, G. W. and W. G. Cochran. 1967. Statistical methods. Iowa State Univ. Press, Ames. 27 Spindler, M. R., Stadler, and H. Tanner. 1984. Amino acid analysis of feedstuffs: determination of methionine and cystine after oxidation with performic acid and hydrolysis. J. Agric. Food Chem. 32:1366. 28 Statistical Analysis System. 1982. ed. SAS Inst., Inc., Cary, NC. 29 Wolff, J. E., E. N. Bergman, and H. H. Williams. 1972. Net metabolism of plasma amino acids by liver and portal drained viscera of fed sheep. Am. J. Physiol. 223:438.