Net Amino Acid Absorption in Steers Fed Alfalfa Hay Cut at Two Stages of Maturity1, 2

Net AminoAcidAbsorptionin SteersFed Alfalfa Hay Cutat Two Stagesof Maturity C. J. SNIFFEN 8 and DON R. JACOBSON Department of Animal Sciences University of Kentucky Lexington 40506

Abstract

Twelve Holstein steers with initial weights of 130 to 202 kg were assigned to a 63-day performance trial on a completely randomized basis for two maturities of alfalfa hay. They were fed at 110% of voluntary consumption. At the end of the performance trial, digestion and nitrogen balance information was obtained on the same steers. Two steers from this group and one additional steer were employed to measure in nine separate 48-h experiments, blood flow by the Doppler shift and telemetry technique and net amino acid absorption from portal-carotid differences. Mean portal blood flow was 39.2 ml/min per kg body weight. Differences in blood flow between treatments were not significant. Advanced hay maturity decreased voluntary intake, feed efficiency, and gain. Further, digestibility of all proximate fractions were lower. Essential, nonessential, and total amino acid absorptions were reduced by advanced maturity to a much greater degree than could l~e attributed to differences in intake of digestible protein. Approximately five times as much amino acid was absorbed from good quality as from reduced quality alfalfa hay.

tures. In addition, digestibility of crude prorein, dry matter, and fiber is reduced as alfalfa matures (4, 8, 12, 35). Associated with increasing maturity of alfalfa hay is a reduction in voluntary feed intake and performance (1, 35). Correlation is negative between dry matter digestibility and advancing maturity, both in vitro and in vivo. The negative correlation between small changes in digestibility and advancing maturity does not predict accurately voluntary feed intake. Appreciation and understanding the quantities and nature of nutrients absorbed and metabolized by the animal fed different qualities of hay would be a more direct and meaningful explanation of differences in voluntary feed intake and performance than the traditional apparent digestion trial and quantitative determination of undigested residue. Some workers have considered quantities of nutrients absorbed and utilized by the host tissue of ruminant animals and have approached the question in a number of ways. These studies include estimates of production of volatile fatty acids, [largely by isotope dilution technique (22)], concentration gradient across the t wall by portal-carotid difference, (especialsugars and volatile fatty acids), disappearance from gut by dual reentrant carmula (7, 36), and measurement of rate of disappearance (1) of chemically defined fractions of alfalfa hay grown in chambers containing radioactive CO2. Because tureen fermentation intervenes between dietary protein and amino acid absorption, it has not been possible to estimate with confidence from dietary amounts, quantities of each amino acid available for absorption in the gastrointestinal tract of the ruminant animal. Weston and Hogan (36) measured disappearance of amino acids from the gut with two qualities of hay by dual reentrant cannula technique. ]acobson et al. (20) have suggested that the protein requirement of ruminants for tissue might best be expressed as "absorbable amino acids." This study was to measure net quantities of each amino acid absorbed into the portal vein when dairy steers were fed alfalfa hay cut at

~y

Introduction

Crude protein is reduced and fiber content is increased in alfalfa hay as the forage maReceived April 15, 1974. The investigation reported in this paper (no. 72-5-86) is in connection with a project of the Kentucky Agricultural Experiment Station and is published with approval of the Director. Data were taken from the thesis submitted to the Graduate School, University of Kentucky by the senfor author in partial fulfillment of the requirements for the Ph.D. degree. ~Present address: Department of Animal and Veterinary Sciences, University of Maine, Orono 04473.

371

372

S N I F F E N AND JACOBSON

two stages of maturity (designated "good quality" and "reduced quality"), and to relate absorption of amino acids to voluntary consumption, rate of gain, and nitrogen balance. ExPerimental Procedure

Performance and digestion trials. Twelve Holstein steers that weighed between 130 and 202 kg were assigned, completely randomized, six each, to receive chopped hay of two stages of maturity. Alfalfa hay of two maturities was fed at 110% of consumption as the only dietary intake except for trace mineralized salt for 63 days. All animals were weighed 3 consecutive days at the beginning and at the end of the experimental period and once weekly during the experiment. At the end of the intake and performance trial all steers were given 100% of their previous ad libitum intake of hay for a 5-day total collection of feces and urine in a digestion and nitrogen balance trial. Appropriate samples of feed offered and refused and of excreta were obtained and analyzed for energy and proximate constituents (2). Nitrogen solubility of forages was measured by methods of Wohlt et al. (37). Data were analyzed in a completely randomized design. Weeks in the performance trial were treated as a split plot. Portal blood flow and absorption of amino acids. Two of the animals in the performance trial and one additional Holstein steer (Table 1) ranging in body weight from 156 to 230 kg and in age from 213 to 322 days were employed in nine separate blood flow and amino acid absorption experiments with the two qualities of alfalfa hay. Two steers received both

treatments with an 18-day adjustment between treatments. During the experiment animals were fed 100% of the previous ad libitum intake. Paired comparisons were between treatments by multiple regression analysis of variance. Times after feeding and diurnal (day vs. night) were treated in a split-split plot analysis. Experiments within animal were used to test the split-split plots. Plasma amino acids were determined by automated analyses (27). Blood flow was measured continuously for two 24-h periods bv the prindple of Doppler shift*. This methocl has been described by Franklin et al. (15) and Can- and Jacobson

(6). Procedures to implant the blood flow transducer and catheters, described in (6), were modified. Instead of inhalation anesthesia by halothane, 2.5% xvlocain was injected into the lumbar processes "for a local anesthetic. The carotid artery was not exteriorized, mad a caecal vein was employed to implant the catheter. The catheters were silicone elastic s with 1.02 mm inside diameter and 1.65 mm outside diameter. The principles of equipment operation have been described (15, 16). To increase the accuracv of measuring blood flow, a high quality stereo frequency modulation receiver was employed instead of a battery operated receiver. Further, to reduce variability, the total velocity signal for any designated period was integratWard Associates, San Diego, CA. 5Dow Co~ing, Midland, MI. 4

T.~nLE 1. Animal allocation and treatment sequence on the two qualities of alfalfa hay during the absorption studies. Treatment Good quality hay Mean Reduced quality hay

Animal

Absorption experiment

649 849 684 684

19 20 22 23

22 22 649 649 684

14 15 16 17 24

Mean * At beginning of experiment. b Dry matter basis. " Grams per body wt. "~. JOURNAL OF DAIRY SCIENCE, VOL. 58, N o . 3

Age" (days) 244 249 265 270 257 315 322 213 222 288 272

Body weight~

Feed intakeb

(kg) 171 189 222 229 203 217 229 156 165 230

( g/BW . . . . ) 131 123 89 92 109 74 85 108 106 87 92

249

373

AMINO ACID ABSORPTION FROM A L F A L F A

ed with an integrating preamplifier 6. At termination of the experiments the animals were sacrificed, and liver and portal vein were excised. The inner diameter of the vessel underneath the transducer was measured by calipers. The following equation was used to calculate flow: Q -- Fd (ID 2) (1.414) (10) -2 where: Q = ml blood/min, Fd ----- Frequency shift in cycles per s, and ID ---- the portal vein inner diameter in mm (15, 16). The system was also calibrated, and flow values were verified by pumping known quantifies of heparinized blood through the portal vein per unit time. Blood flow was then regressed on frequency shift. Absorption was determined by this equation: At = (Pt -- Ct)Bt(1-H) where: A t ---- net absorption for time t, Pt = portal plasma concentration during time t, C t ---- carotid plasma eoncentration during time t, B t -- blood flow in liters during time t, and (l-H) = mean red blood cell concentration during the same time interval. Total net absorption was determined by summing four sampling times over 24 h. Blood samples were taken at 2-h intervals after feeding, and three consecutive 2-h samples were pooled for analysis for each of the four 6-h intervals. Results

Proximate composition, gross energv, and amino acid content of regrowth alfalfa hay cut at two stages of maturity (July 6 and July 21) from the same field are in Table 2. Protein was about 5% higher and fiber was 7% lower in the good quality hay (cut July 6). Total amino acids decreased, and nitrogen solubility increased with advancing maturity. Portal blood flow of the steers is in Table 3. Blood flow was similar on the two qualities of hay, diurnally and hours after feeding. The mean was 39.2 ml/min per kg body weight. When blood flow (liters/h) was regressed on body weight (kg) the following relationships were evident: Y ---- 2.42 X --13.12 and Y = 3.05 X --128.9; with correlation coefficients of .82 and .98 on good and reduced quality hays * Hewlett Packard, Waltham, MA.

TABLE 2. Amino acid and proximate composition of alfalfa hay harvested at two different maturities a. Constituent b Dry matter (~; o~ feed) Crude protein Ether extract Crude fiber Ash Gross energy (kcal/gm) Nitrogen solubility Lys His Arg Thr Val Met Ite Len Phe Cys Asp Ser Glu Pro clv Ala Tyr Total AA

Goodc

l~educed n

91.2 18.8 3.1 27.5 9.9 4.4 26.2 .71 .23 .69 .62 .94 .06 .72 1.23 .85 .04 1.61 .50 1.63 .89 .84 .91 .38 12.85

92.4 13.9 2.6 34.8 9.0 4.3 32.7 .53 .17 .44 .43 .79 .15 .56 .86 .60 .20 1.21 .37 1.10 .62 .61 .74 .26 9.64

Dry matter basis. b Percent basis. Proximate composition meart ~ 10 samples; AA mean of 2 samples. d Proximate composition mean of 5 samples; AA 1 analysis. where mean body weights were 206 and 184 kg. Regressing flow on body weight (kg -rn) did not improve correlations. All differences in analysis of variance for regression slopes, treatments, postprandial, and diurnal effects were nonsignificant. Plasma amino acid concentrations. Concentrations of essential and nonessential amino acids on both treatments are in Tables 4 through 7. Except for aspartic acid all daily mean concentrations of portal plasma amino TABLE 3. Portal blood flow in steers fed two qualities of alfalfa hay. Hours after feeding Day Night Date cut Day " 0-6 6-12 0-6 6-12 July 6

1 2 July 21 1 2

40.3" 41.4 39.4 40.0

(ml/min/kg) 38.6 40.0 38.9 41.4 40,8 40.2 38.0 38.7 39.0 39.4 38.6 38.2

SD ___3.4 +__3.0 +__3.8 +3.1

" Nine observations per mean. JOURNAL OF DAIRY SCIENCE, VOL. 58, No. 3

374

SNIFFEN A N D

JACOBSON

TABLE 4. Plasma essential amino acids in steers fed good quality hay cut July 6. Diurnal

Hour after feeding 0-6 6-12

Amino acid

Vessel"

Meanb

Day

Night

Lys

P C

309 209

359 207

(urrt/1) 243 212

336 234

SD"

282 184

172 69

P

125

158

81

169

81

143

C

84

82

86

100

68

27

Arg

P C

170 119

179 111

159 128

168 129

171 108

62 53

Thr

P C

379 296

312 315

468 271

344 331

415 262

231 188

Val

P C

799 628

684 545

952 739

867 638

731 618

403 344

Met

P C P C P C

61 40 322 228 409 275

57 26 283 208 331 232

66 59 373 256 513 332

63 46 360 233 468 285

58 35 284 223 350 265

23 30 145 103 281 119

P C

158 126

124 126

202 126

179 114

136 137

68 102

H~

Ile Leu Phe

P = portal, C = Carotid. c Variation includes animal, diurnal and hours after feeding. b Four animals, four periods each 24 h. acids were higher on good quality hay. Carotid concentrations, except for eystine, ]ysine, histidine, and arginine, were all higher on good quality hay. Standard deviations of portal con-

centrations on good hay were generally larger than those of the carotid artery. This trend is not evident in the steers fed reduced quality hay.

TABL~ 5. Plasma nonessential amino acids in steers fed good quality alfalfa hay cut July 6. Diurnal

Hour after feeding 0-6 6-12

Amino acid

Vessel"

Meanb

Day

Night

Cys

P C

60 29

83 35

(urn/l) 29 22

69 34

51 25

104 27

Asp

P C P C

41 61 283 178

39 53 248 154

44 70 328 208

34 85 250 213

48 30 315 142

20 68 147 105

P C P C P

211 168 175 207 594

193 159 161 257 509

235 179 193 141 706

218 159 180 212 539

203 176 169 203 649

85 90 75 151 198

Set Glu Pro Gly

SD~

C

429

403

463

431

427

105

Ala

P

647

612

293

677

616

296

Tyr

C P C

316 137 97

295 110 93

344 174 103

338 149 99

295 126 95

112 64 53

P ~- Portal, C --'--Carotid. ~ Variation includes animal, diurnal and hours after feeding. b Four animals, four periods each 24 h. JOURNAL OF DAIRY SCIENCE, VOL. 58, NO. 5

AMINO ACID A B S O R P T I O N F R O M ALFALFA

375

Ta.BLE 6. Plasma essntial amino acids in steers fed reduced quality alfalfa hay cut July 21. Diurnal

Hour after feeding 0-6 6-12

Amino acid

Vessel"

Mean b

Day

Night

SD ~

Lys

P C

263 242

224 231

250 256

243 272

283 212

92 91

His

P C

1@2 103

102 114

101 89

102 110

102 96

35 33

Arg

P C

169 159

181 157

153 161

160 169

178 149

58 44

Thr

P C

167 155

176 132

156 184

179 153

156 151

93 113

Val

P C

304 259

322 257

282 263

299 305

310 214

45 80

Met

P C

37 35

41 37

31 33

34 47

39 23

16 35

Ile

P C

154 128

158 119

148 138

145 145

160 110

45 37

Leu

P C

191 163

193 151

187 177

192 185

190 141

48 44

Phe

P C

79 81

80 66

78 101

72 79

87 84

30 62

(urn/l)

P = Portal, C = Carotid. b Five animals, four periods each 24 h. Includes animal, diurnal and hours after feeding. Differences in concentrations between day and night of essential amino acids (Table 4) and nonessential amino acids (Table 5) in the

steers f ed good quality hay were not consistent, but when we compared 0 to 6 vs. 6 to 12 h after feeding there was a trend suggesting

T.~LE 7. Plasma nonessential amino acids in steers fed reduced quality alfalfa hay cut July 21. Diurnal

Hour after feeding 0-6 6-12

Amino acid

Vessel"

Mean b

Day

Night

SD ~

Cys

P C

29 39

33 57

2,4 17

24 43

34 36

22 49

Asp

P C

43 24

33 21

55 98

40 23

46 27

37 15

Ser

P C

127 133

128 121

127 149

113 126

142 140

42 60

Glu

P C

132 144

125 103

140 195

136 136

127 153

53 103

Pro

P C

84 82

84 68

85 101

85 81

84 84

34 44

Gly

P C

357 308

355 289

360 33~

334 295

381 321

31 35

Ala

P C

264 221

281 196

243 254

247 213

282 231

41 48

Tyr

P C

65 55

69 50

60 60

60 62

70 47

23 23

(urn/l)

"P = Portal, C ----Carotid. b Five animals, four periods each 24 h. Variation includes animal, diurnal and hours after feeding. JOURNALOF DamY SCIENCn,VOL. 55. NO. 3

TABLE 8. Net amino acid absorption in steers fed good or reduced quality alfalfa hay. ~" Quality of hay

Good

24 h Reduced

Diurnal Good SD ~

z Amino acids Essentml Lys H~ Arg Z 9 Thr Val Met Ile Leu Phe TotM Non e~enfial Cys

Day

Night

Reduced Day Night

Good 0-6

Hour after feeding Reduced 6-12 0-6 6-12

( m g / h / B W '~) 94 44 54 **b 51" 128 15" 78 180"* 37

9 3 2 14 38 6 27 35 5

157 75 30 171 197 11 70 129 90

141 79 73 7 105 30 64 162 4 *¢

47 10 35 109 152 --1 90 198 71

12 --11 --1 29 48 2 33 35 15

5 16 6 --7 29 9 21 36 --5

90 68 42 17 171 15 100 217 65

681"*

138

342

651

712

167

108

--21

--39 9 6 21

29

20

4

46 1

66 85 86 15 56 143 9

15 24 18 7 15 25 4

19 4 59 18 38 46 6

785

578

39

236

4 19 5 --29

63 --35 33 61

57 9 102 35

--27 14 1 12

--16 14 10 19

ii

--6

--21

--ii

6

-- 1

17 31 19

58 189 52 17

119 160 35 14

25 45

66 10

30 50 22 19

22 36 24 25

148

79

41

Asp

--13

14

40

--13

--12

Set Glu Pro Gly

5 6 3 24

50 100 49 101

41

109

Tyr NI-Ld

68** 48 -- 16 88 174" 43** 16

16 19

20 5

Total

453*

77

338

325

582

109

46

400

507

87

69

216

651

976

1,287

276

154

1,185

1,084

126

305

Ala

Total

60

98

1,135"*

.4 Square root of the main plot error mean square. b * (P<.05), ** (P<.Ol). c Significant diurnal by treatment interaction. a Not included in totals.

59 29 - - 7 8 *° 48 207 20 *~ 21

76 66 45 128 171

ii

i0

15

o :z

377

AMINO ACID ABSORPTION FROM ALFALFA

h 0

10

"%~ ~ l ~

Good Quality

"~"

------ Reduced'Quallty

0

AMINOACID

Fro. Z. Portal arn~o acids from two qualit~e~ of alfalfa hay fed to Holstein steers. Each point represents the mean molar percentage of a specific amino acid of the total amino acid analyzed. Amino acid ranking is based on good quality hay.

higher plasma concentrations at 0 to 6 h. This trend was reversed on the reduced quality hay. Plasma essential amino acids (Table 6) and nonessential amino acids (Table 7) in steers fed reduced quality alfalfa hay tended to be higher at 6 to 12 h after feeding. For individual portal plasma amino acid concentrations as a percent of the total concentration, the mean molar concentrations as a percent of the total molar concentration are in TABLE 9. Amino acids consumed and absorbed in Consumed~ Amino acids Good Reduced

Fig. 1. In determining ranks of amino acids, the mean portal plasma concentrations for good hay were used in comparison. There are trends suggesting that valine, alanine, and leucine were lower in concentration on reduced quality hay whereas glycine, lysine, arginine, and histidine were higher. Amino acid absorption. Net amino acid absorption in steers fed good or reduced quality hay is in Table 8. Animals fed good alfalfa hay absorbed arginine, threonine, methionine, leucine, serine, alanine, and tyrosine at significantly higher rates. The greater individual absorption rates on good hay resulted in total essential, total nonessential, and total amino acids being absorbed at significantly higher rates. Total amino acids were absorbed from good quality hay at five times the rate from reduced quality hay. Generally diurnal amino acid concentrations in Tables 4 through 6 are reflected in absorption rates. Animals on good quality hay absorbed amino acids at a greater rate at night than during the day. On reduced quality hay the reverse was usually true. In the cases of phenylalanine, proline, and tyrosine, interaction was significant (P < .05). steers consuming good or reduced quality hay. Net absorbed Absorbed/consumed Goodb Reduced * Good Reduced

( mg/dayfBW.,5 a) Essential Lys His Arg Thr Val Met Ile Len Phe Total Non essential Cys

775 253 754 679. 1,020 63 784 1,339 929 6,589

493 161 405 393 734 134 512 791 553 3,771

2,252 1,186 1,2.99 1,214 3,085 353 1,864 4,328 897 16,350

208 62 48 322 916 129 652 852 129. 3,311

291 469 173 181 309. 560 237 323 97 248

42 39 12 82 125 96 127 108 22 88 --278 30 37

42,

181

1,447

-- 504

3,445

1,749

1,112

-- 306

336

-- 17

Set 546 344 Clu 1,769 1,010 Pro 968 572 Cly 914 562 Ala 984 680 Tyr 417 242 Total 7,389 4,703 Total 13,979 8,474 Dry matter basis. b Mean contains four observations. Mean contains five observations. o Milligrams per day per body wt '7~.

1,624 1,151 -- 388 2,125 4,181 1,038 10,879 27,230

129 --86 57 565 972 400 1,852 5,170

297 65 -- 40 232 425 249 147 195

Asp

--9 I0 i01 143 165 39 61

JOURNAL OF DAIRY SCIENCE, VOL. 58, NO. 3

378

SNIFFEN AND JACOBSEN

TABLE 10. Performance of steers fed two qualities of alfalfa haya.

Trait

Treatments Good Reduced quality quality

Units

Dry matter intake Dig e energy intake Dig protein intake Dig energy/dig protein Avg daily gain Feed/gain Dig energy/gain Dig protein/gain

kg/100kgBW c kcal/kgBW g/kgBW kcal/g g/day kg/kg Mcal/kg g/kg

3.0 54.7 2.6 21.1 906 5.91 15.4 738

2.6 42.9 1.6 27.6 597 7.31 17.5 632

Overall u mean

SD

2.82 *d 48.8** 2.1"* 24.4*** 751"* 6.61" 16.4 685

-+.60 ---7.5 ±.6 ± 3.3 ± 131 ---2.1 ±5.0 ±221

* Fed 10~ in excess of ad libitum intake. b Six observations per mean. c Body weight. d *P~.05; **P~.01. e Digestible. Though not sign~cant, trends suggested the performance trial are in Table 10. Steers that total amino acids were being absorbed at on good quality hay consumed more hay (P a greater rate 0 to 6 h after feeding on good < .05), digestible energy (P < .01), and diquality hay "whereas the reverse was true for gestible protein (P < .01). This resulted in the reduced quality hay. Further, the nones- higher gains (P < .05) and lower amounts of sential rate was greater at 6 to 12 h on the feed required per unit gain (P < .05). The keal digestible energy consumed/g digood hay. Absorption as a percent of consumption is gestible protein consumed was lower (P < in Table 9. Animals on good quality hay con- .01) for animals on good quality hay. Though not significant, the higher digestion sumed about 1.6 times more amino acids than those on reduced quality hay. In contrast, they coefficients (Table 11) on the good quality absorbed 4.9 times as much of the essential hay corroborate performance data. Further, amino acids and 5.9 times as much of the non- there was an average of 24 g more nitrogen or essential amino acids or 5.3 times as much of 150 g of protein retained daily on good quality hay. In almost all cases there were greater (P the total amino acids. Animals on good quality hay absorbed 248% < .05) intakes of aft proximate fractions. of the essential amino acids consumed as comDiscussion pared to 85% on the reduced quality hay. Blood [low. Mean blood flow was well withThey also absorbed 147% of nonessential amino acids and 195% of total amino acids as in the range of flows in this laboratory and recompared to 39 and 61% on reduced quality ported by other workers (5, 6, 17, 19). The experimental variation as a percent of the hay. Performance and digestion. The results of mean (39.2 ml/min per kg body weight) was TABI~ 11. Digestibility eoefllcients of two qualifies of alfalfa hay fed to steers a. Treatment

DMb

Energy

Protein

Good= Reduced~ Mean SD

62.1 58.2 80.0 4.5

62.1 55.5 58.5 5.8

66.5 62.2 64.1 7.8

EE c

Fiber

NFE d

62.4 48.5 54.8 13.1

55.2 52.1 53.5 4.8

68.1 64.8 66.3 5.2

(%)

* Fed at 100% of ad libitum. b Dry matter. Ether extract. Nitrogen free extract. Nitrogen balance. Six observations per mean. g Five observations per mean. JOURNAL OF DAIRY SCIENCE, VOL. 58, NO. 3

N Bale (gm/day) 31.9 7.9 18.8 26.7

AMINO ACID ABSORPTION FROM ALFALFA

13.8%. Variation within animal was ___ 1.6 ml/min per kg body weight, which was low. It demonstrates the repeatability of the technique and that there was little postprandial effect. Bensadoun and Reid (5) and Hume et al. (19) with sheep reported a significant postprandial effect. There appears to be a specie and perhaps diet difference. When blood flow was regressed on body weight, there was not a significant difference between slopes for the two treatments. Under conditions as in these experiments blood flow could be estimated with accuracy without the necessity of measuring blood flow per se. Amino acid concentrations. The higher portal plasma concentrations of amino acids on good quality hay was influenced by diet. The same trends were true for carotid concentrations. However, concentrations of cystine, lysine, histidine, and arginine were higher for animals on the reduced quality hay. Rogers and Harper (33) studied plasma amino acid concentrations concurrently with tissue pools and found that changes in plasma amino acid patterns appeared to reflect changes that occurred in muscle. They felt that the plasma pool might be helpful in determining limiting amino acids. Christensen (11) in the same symposium discussed problems of interpretation of plasma amino acid concentrations. He postulated several operating transport systems which influence cell uptake of amino acids. These transport systems differ mainly according to chemical properties of the amino acids; some systems concentrate or transport amino acids faster. Fig. 1 is a study of relative concentrations of amino acids and suggests a dietary influence. Further, Snyderman et al. (34) corroborated that where there are deficiencies or excesses of amino acids, other amino acids tend to be affected as a group. The phenomena appears in ruminant animals. There is a suggestion that amino acid imbalances can occur on standard rations. One might find the conditions demonstrated here even more pronounced in a lactating cow where demands can be even greater on the amino acid pool (20). There were large variations in plasma amino acid concentrations (Tables 4 through 7) which increase the difficulty of interpreting the data. Many authors including Weston and Hogan (36) have demonstrated that amino acid concentrations in peripheral blood are variable. Weston and Hogan (36) found that advancing maturity of hay increased peripheral plasma lysine, histidine, and arginine concentrations.

379

In this study the same amino acids from the carotid were only 86, 82, and 75% as concentrated for the good hay. However, the reverse was true for portal samples. One possible explanation is by Christensen (11), who demonstrated that these acids are in the same transport system, and Snyderman et al. (34) have shown that when one amino acid is imbalanced, several other acids will be affected usually within a system correlating fairly closely to their chemical and physical properties. Another explanation is the increased relative utilization of these amino acids on the good hay (Table 13). Diurnal and postprandial variation can be important for not only deciding sampling frequency but giving insight into possible mechanisms of absorption. There is little to suggest the cause of the generally higher portal concentrations at night on good hay as compared to reduced quality hay. It may be associated with the gastrointestinal retention time of the particular hay. The postprandial total amino acid data suggest that there is increased absorption at 0 to 6 h after feeding a good quality hay. This trend was reversed on reduced quality hay. Amino acids absorbed on good hay might be more heavily influenced by dietary amino acids and also might reflect differential postprandial rates of passage. Absorption. Diurnal and postprandial interactions (Table 8) that are mediated by the diet are suggested by trends. There are not adequate explanations for the diurnal absorption rates; however, the postprandial absorption interactions might be caused by differences in rumen bypass mediated by protein solubility and/or by rate of passage. To amplify further on the absorption patterns, simple correlations of amino acids absorbed were determined (Table 12). There were 39 statistically significant correlations. The correlations among valine, leucine, and isoleucine were the highest (mean of .84). The mean correlation of alanine with the above amino acids was .79, of serine with the same amino acids was .61, of tyrosine with the same was .60, of glutamic acid and glycine with the same were both .49. These correlations together with those that remained among the previously mentioned amino acids were significant except two and accounted for all but 13 of the 39 total. Correlations with phenylalanine accounted for four of these. Arginine was significantly correlated with five amino acids including lysine. The only amino acid correlated with lysine was arginine. Histidine, beJOURNAL OF DAIRY SerENelY, VOL. 58, NO. 3

O

CO O

o

g~ TABLE 12. Simple correlations of amino acids absorbed at six-hour intervals a.

Lys His Arg Thr V~ Met Ile Leu Phe NH8 Cys Asp Set Glu Pro ~ly Ala Tyr

Lys

His

Arg

1.00 .05 .42 .09 .04 .21 .23 .07 .12 .35 .40 .16 .18 .17 .16 .15 .28 .20

1.00 .35 .04 .09 .27 .10 .03 .21 .07 --.01 .14 .07 .26 .41 .08 .11 .24

1.00 ,21 .30 .31 .46 .29 .17 .23 .12 .07 .42 .41 --.07 .35 .46 .51

Tilr

1,00 --.06 .23 .05 .08 .08 --.19 --.06 .26 .47 .45 .18 .33 .26 .28

Val

Met

1.00 --.05 .83 .88 .39 --.13 .10 .16 .60 .48 .29 .58 .76 .56

1.0O .11 .04 .12 --.09 .13 --.04 .20 .00 --.23 --.18 .18 .23

lie

Leu

Pbe

NH.~

Cys

Asp

Ser

Glu

Pro

Gly

Ala

Tyr

z J 00

.82 .58 --.04 .68 ,12 .50 .52 .35 .46 .75 .72

1.00 .33 --.16 .01 --.03 .72 .48 .34 .44 .86 .52

z 1.00 .08 .07 .00 .19 .49 .12 .26 .46 .70

1.00 .18 --,09 --.12 .07 --.09 --.17 --.01 --.01

1.00 .02 --.08

.03 --.24 .14 .10 .16

1.00 .34 .26 .11 .48 .10 .00

oc3 1.00 .60 .33 .58 .80 .37

1.00 .41 .64 .58 .44

1.00 .39 .12 .18

1.00 .47 .41

1.00 .58

1.00

All values which exceed .41 are significant at P < ,05 and all values which exceed .53 are significant at P < .01. Absorption expressed as g/day per unit metabolic size.

381

AMINO ACID ABSORPTION FROM ALFALFA

TABLe. 13. Comparison of essential plasma amino acid concentrations and absorption in animals fed two qualities of alfalfa hay. Amino acid

Hay quality

Portal S

Arg

Good Reduced Good Reduced Good Reduced Good Reduced Good Reduced Good Reduced Good Reduced Good Reduced Good Reduced

170 169 125 103 158 79 61 37 309 263 379 162 322 154 409 191 799 304

His Phe Met Lys Thr lie Leu Val

Concentration Carotid a P/C b P ( G/R ) ~

(ttm/1).-119 159 84 102 126 81 40 35 200 242 296 155 288 128 275 163 628 259

143 106 148 101 125 98 153 106 148 109 128 108 141 !20 149 117 127 117

(%) 101 121 200 165 117 234 209 214 263

C ( G/R )'l

Daily absorption R/G e

(mg/day/BW ~*) 1,299 75 48 1,186 82 62 897 156 122 353 114 129 2,252 86 208 1,214 191 322 1,864 178 652 4,328 169 852 3,085 242 916

(g) 4 5 14 36 9 26 35 20 30'

Arithmetic mean. b Portal/carotid. Portal concentration, good hay/reduced quality hay. a Carotid concentration, good hay/reduced quality hay. e Absorption on reduced quality hay as $ of that on good quality hay. f Mflligrmns per day per body wt "75. Absorption based on the sum of four portal/carotid differences times the blood flow for each interval. cause of its variable net absorption pattern in this study, was not sigrtificantly correlated with any amino acid but approached significance with arginine. These observations are confirmed, in part, by the work of Christensen (11), who postulated that there are several systems wherein amino acids act as a group. For example, he described one system (L-system) that includes those amino acids with apolar, branched side chains and aromatic rings. It includes particularly valine, leucine, isoleucine, and the amino acids with aromatic groups. In Table 13 not onlv are carotid plasma lysine, histidine, and argini'ne concentrations increased with advancing maturity but leucine, threonine, isoleucine, and valine are depressed. Absorptions of all of these amino acids except threonine have been highly correlated (Table 12) and appear to be in the same transport systems (20). Increases of these amino acids suggest among other possibilities competition for absorptive sites not only from the gut but also into the tissues. This possibility is partially substantiated by the higher daily absorption (Ta-

ble 13) of leucine, isoleuclne, and valine on the reduced quality hay when they are a percentage of the total. Correlations in Table 12 suggest that there are two transport systems, one for lysine, arginine, and histidine, and one for leucine, isoleueine, and valine. Correlation of amino acids between the two groups was generally quite low. It is postulated that one group might saturate the absorptive sites not only of its own transport system but also of the lysine system. This phenomenon has been shown by Snvderman et al. (34). It is possible with amino acid absorption in ruminants to have not only imbalances but also to have situations where there can be competition for active transport sites, In Table 14 the microbial and hay amino acid patterns are presented for comparison to daily absorption patterns, The most striking differences in the essential amino acids are the high valine and leucine percentages on both hays and the low lysine, histidine, and arginine on reduced quality hay. Hume et al. (18, 19) in similar comparisons on alfalfa hay in sheep fed at 2-h intervals did not observe these differences. Their absorption patterns were simiJOURNAL OF DAIRYSCIENCE, VOL. 58, No. 3

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SNIFFEN AND JACOBSON

TABLE 14. Ratios of absorbed, consumed and microbial amino acids. Amino acids

Daily absorption" Good Reduced

Microbial b Bacteria Protozoa

Hay consumed~ Good Reduced

(%) Essential Lys His Arg Thr Val Met Ile Leu Phe Total Nonessential Cys Asp Set Glu Pro Glv Ala Try Total

8.3 4.3 4.8 4.5 11.3 1.3 6.8 15.9 3.3 60.0

4.0 1.2 .9 6.2 17.7 2.5 12.6 16.5 2.4 64.0

9.3 2.3 5.4 5.5 6.6 2.6 6.4 7.3 5.1 50.5

10.1 2.3 4.9 5.1 5.2 2.2 6.9 8.1 6.2 50.8

5.6 1.8 5.1 4.8 7.4 .5 5.6 9.6 6.6 47.0

5.6 1.8 4.6 4.4 8.3 1.5 5.8 8.9 6.2 41.1

5.3 -- 1.1 6.0 4.2 -- 1.4 7.8 15.3 4.0 40.0

--9.8 6.5 2.5 -- 1.7 1.1 10.9 18.8 7.7 36.0

1.0 11.1 3.8 11.9 4.1 6.1 6.5 4.2 49.5

1.0 12.4 3.6 12.5 3.7 5.0 5.2 5.4 49.2

.3 12.6 3.9 12.6 6.9 6.6 7.1 3.0 53.0

2.0 12.5 3.9 11.4 6.4 6.3 7.7 2.7 52.9

Derived from 24-h data in Table 8. b Purser and Bueehler ( 32 ). c Dry matter basis. lar to amino acid patterns in bacteria (32) and in hay. In recent work Mangan (24) quantitated the release and metabolism of amino acid in the rumen. He found that at I h after administration of casein into the rumen, there was a large increase in valine, leucine, isoleucine, and lysine. He suggested that there was synthesis in addition to degradation. The high proportions continued over 1.5 h. Several amino acids exhibited negative absorption rates (Table 8). There are two reasons for these phenomena. First, there may be little of the amino acid being presented for absorption. This coupled with utilization by the gut and change of one amino acid into another can produce low or negative net absorption rates. Of the nonessential amino acids, glyeine, alanine, and tyrosine were absorbed in unusually high amounts as compared to those consumed (Tables 9 and 13). Hume et al. (19) in this laboratory noted the same trends in sheep fed alfalfa hay. It was partially explained as extensive transamination of glutamic and aspartic by the gut wall. It is startling to find such a contrast in absorption of amino acids between the hays of two maturities. Part of the contrast can be exJOURNAL OF DAIRY SCIENCE VOL. 58, NO. 3

plained by the difference in the amount of protein consumed or digested. However, when absorption is proportionally adjusted to the same intake, steers on good hay still absorbed three times more amino acids than steers on reduced quality hay. Explanations for the absorption contrasts are not apparent. Weston and Hogan (36) studied the effects of hays of two maturities on intake and digestion employing external and internal markers. The digestion trials corroborated our studies. Further, they found that nitrogen balances and plasma amino acid concentrations were greater than could be attributed to differences in protein intake. They did study this unusual event further by feeding the subterranean clovers of two maturities on an isonitrogenous basis and found the same phenomena. In similar studies with Berseem clover they did not observe the same relationships, suggesting a species interaction. Coelho Da Silva et al. (7) studied amino acid absorption sites in sheep as affected by the physical form (chopped, wafered, and pelleted) of the diet (lucerne hay). They found that amino acids being presented at the proximal duodenum ranged from 100 to 50% in excess of what was ingested. They suggest that mic~0bial fermentation enhances amounts of

AMINO A C I D A B S O R P T I O N F R O M A L F A L F A

some amino acids entering the small intestine, namely, methionine, lysine, tryptophan, and eystine. Methionine, lysine, cystine, and others were absorbed in considerable excess of what was consumed (Table 9). The digestion and performance trials, though demonstrating significant differences between treatments, do little to suggest the large contrasts in absorption. From T~ble 9 animals on good quality hay consumed 14,560 mg of digestible protein/ day per kg body weight -zS. The absorbed:consumed ratio for total amino acids became 1.90. Animals on reduced quality hay consumed 8,170 mg of digestible protein/day per kg body weight .rS. As a ratio of absorbed, it became .63. This compares favorably with 195 and 61% for the good and reduced quality hay. Since digestible protein is confounded by endogenous protein, we compare the total absorbed amino acids in terms of crude protein ingested. Animals on good hay consumed 21,890 mg crude protein/day per kg body weight .~5 yielding a ratio of 121%. The protein consumed by the animals on reduced quality hay was 12,140 rag/day per kg body weight .rs yielding a ratio of .39. The reduced quality absorption is similar to the value of Hume et al. (19) (.50) but far below the good quality hay. Animals on good quality hay absorbed more amino acids than they consumed. In terms of crude protein intake, animals on good quality hay absorbed three times more amino acids per gram of protein consumed than did the animals on poor quality hay. To account for the high rates of absorption on the good quality hay there are several possibilities. It is conceivable that a high proportion of dietary protein was absorbed. Mason (25, 26) suggested little dietary nitrogen in the feces. Further, he demonstrated that a high percentage of fecal nitrogen was of bacterial origin. Even ff 100% of the dietary crude protein was utilized, there would be at least 21% more of the absorbed amino acid to account for. This likely would be accounted for by increased microbial output (7) through recycling of nitrogen to the rumen via the rumen wall and saliva and especially through the endogenous secretions (18, 19, 28). If one considers, however, dietary amino acid degradation in the rumen and incomplete digestion and utilization of microbial protein, a great deal of recycling of amino acids from the gut lumen to the portal

383

vein and ultimately back to the gut lumen occurred. This was true under conditions of a high protein intake and high concentration of amino acids in the portal vein such as for the good alfalfa hay. The same hay cut at a later date greatly reduced absorption of amino acids, only 61% of amino acid consumption. Apparently, recycling of amino acids was greatly reduced. Perhaps dietary protein was not utilized as efficiently. There was a lower nitrogen digestion coefficient and higher NH 3 absorption rate to lend credence to this. There could have been a combination of increased rumen retention and reduced protein solubility as well. Further, there is the possibility that the amino acids were not in the right ratios or amounts to stimulate microbial protein output (29). Another factor might be the inefficient utilization of NHa by bacteria. It is also possible that the amino acids were not in the right ratios at the absorptive sites (Fig. 1 and Table 12) resulting in lower uptakes (19, 29, 34). We relate absorbed amino acid to gain and protein retained as measured in the performance and digestion trials. One and six-tenths g vs..46 g of amino acids were absorbed for each gram of gain in body weight on the good and reduced quality hay. In terms of protein retained (g N X 6.25) the values were 7.4 and 5.6. There was a slightly more efficient utilization of dietary protein on the reduced quality hay diet. This conforms to the performance trial where 738 and 632 g of digestible protein per kg of gain were consumed on good and reduced quality hays. Voluntary feed intake. Various reasons have been proposed for the adverse effect increasing maturitv has on intake (3, 9, 10, 13), especiallv the theory of protein nutrition (14). In Table 10 animals on good quality alfalfa hay consumed more protein (P < .01). Egan (14) noted increases of feed intake when nitrogen concentration increased in the ration. Further, he demonstrated that, upon addition of casein per duodenum, feed intake also increased. Peng and Harper (31) and Leung and Rogers (23) found that amino acid imbalances might be related to food intake. Data in Table 11 and Fig. 1 suggest that not only the total protein status of the animals but the ratio of amino acids and their apparent absorption characteristics might be implicated. Limiting amino acids. Under certain conditions individual amino acids may become limiting (19, 29) in ruminant diets. Comparisons between amino acid concentrations and amino acid absorption are in Table 14. In shortest JOURNAL OF DAIRY SCIENCE VOL. 58, NO.

384

SNIFFEN AND JACOBSON

supply on the good hay would be methionine and on the reduced hay, histidine. Snyderman et al. (34) demonstrated individual amino acids have profound effects on other amino acids. If these interrelationships are accepted, not only is the amount of one amino acid important, but more important is the relation to other amino acids. Lysine, histidine, and arginine were all in higher carotid concentration on reduced quality hay. This could reflect inefficient utilization of the amino acid due to low concentration of other amino acids, possibly valine, alanine, Ieucine, threonine, and isoleucine (Fig. 1). To gain more insight into possible limiting amino acids we summarized net daily absorption data (Table 13). On reduced quality hay arginine and histidine were absorbed least with valine being absorbed in the greatest amount. If, however, absorption on reduced quality hay is expressed as a percent of good quality ha)" then arginine, histidine, and lysine were absorbed in small amounts as compared to good quality hay. Methionine and phenylalanine though absorbed in small amounts were not depressed pereentagewise to the extent that the mentioned amino acids were. If the optimum ratio and amount of amino acids absorbed into the portal vein for a given function were known, measurement of absorption would reveal those first limiting.

(4)

(5)

(6)

(7)

(8)

(9)

(10)

Conclusion

For alfalfa hay, increased maturity adversely affects digestibility and performance. Corrected for differences in protein intake, it has a pronounced effect on daily amino acid absorption. The absorption results could be interpreted to mean that there are factors such as protein quality and solubility which under certain conditions play a much larger role in ruminant protein nutrition than has been supposed before. Further, the described technique might be further developed as a method for not only determining limiting amino acids for ruminants but also as a method for determining the amino acid requirement of ruminants. References

(1) Alexander, C. L., tL M. Meger, and E. E. Bartley. 1969. Rumen removal rates of some chemically defined fractions of ~4C-labeled alfalfa. 1. Anita. Sci. 29:746. (2) Association of Official Agricultural Chemists. 1960. Official methods of analysis A.O.A.C. 9th ed. Washington, DC. (3) Baile, C. A., and I. Mayer. 1968. Effects of intravenous versus intraruminal iniections JOURNAL OF DAIRY SCIENCE, VOL. 58, N o . 3

(11)

(12)

(13)

(14)

(15)

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AMINO ACID ABSORPTION FROM A L F A L F A

(16) Franklin, D. L., D. E. Watson, K. E. Pierson, and R. L. VanCitters. 1966. Technique for radio telemetry of bloodflow velocity from unre..trained animals. Amer. ]. Meal. Elec. 5:24. ( 17 ) Fries, G. F., and G. H. Conner. 1961. Studies on bovine portal blood. II. Blood flow determinations with observations on hemodilution in the portal vein. Amer. J. Vet. Res. 22:487. (18) Hume, I. D. 1970. Synthesis of microbial protein in the tureen. IlL The effect of dietary protein. Aust. J. Agr. Res. 21:297. (19) Hume, I. D., D. R. ]acobson, and G. E. Mitchell, ]r. 1972. Quantitative studies on amino acid absorption in sheep. J. Nutr. 102:495. (20) Jacobson, D. R., H. H. VanHorn, and C. J. Sniffen. 1970. Lactating ruminants. Fed. Proc. 29:35. (21) Leibholz, ]. 1971. The absorption of amino acids from the rtunen of the sheep. I. The absorption of amino acids from sohltions in the washed ~amen in vivo. Aust. J. Agr. Res. 22: 639. (22) Leng, R. A., and G. J. Leonard. 1965. Measurement of the rates of production of acetic, propionie and butyric acids in the rumen of sheep. Brit. J. Nutr. 19:469. (23) Leung, P. M. B., and Q. R. Rogers. 1971. Importance of preyrffmTa cortex in foodintake response of rats to amino acids. Amer. ]. Physiol. 221:929. (24) Mangan, ]. L. 1972. Quantitative studies on nitrogen metabolism in the bovine tureen. Brit. J. Nutr. 27:261. (25) Mason, V. C. 1969. Some observations on the distribution and origin of nitrogen in sheep feces. J. Agr. Sci. 73:99. (26) Mason, V. C. 1971. Some preliminary observations on the nature of factors; influencing the excretion of non-dietary faecal nitrogeaa by ruminant animals. J. Agr. Sei. 76:157. (27) Moore, S. D., H. Spackman, and W. H. Stein. 1958. Chromatography of amino acid~ on sulfonated polystyrene resins; An

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improved system. Anal. Chem. 30:1185. (28) Nolan, I. V., and R. A. Leng. 1972. Dynamic aspects of ammonia and urea metabolism in sheep. Brit. ]. Nutr. 27:177. (29) Orskov, E. R., C. Fraser, and E. L. Corse. 1970. Tile effect on protein utilization of feeding different protein supplements via the rumen or via the abomasum in young growing sheep. Brit. J. Nutr. 24:803. (30) Oser, B. L. 1965. Hawks physiological chemish3', 14th ed. McGraw-Hill, New York. (31) Peng, Y., and A. E. Harper. 1970. Ami:lo acid balance and food intake: Effect ot different dietary amino acid patterns on the plasma amino acid patterns of rats. J. Nntr. 200:429. (32) Purser, D. B., and S. M. Buechler. 1966. Amino acid compositiort of hydrolysates of microbial preparations from the rumen of sheep. Aust. J. Biol. Sci. 10:384. (33) Rogers, Q. R. and A. E. Harper. 1968. Significance of tissue pools in the interpretation of changes in plasma amino acid concentrations. Pages 107-126 in Protein nutrition and free amino acid patterns. I. H, Leathern, ed. Rutgers University Press, New Brunswick, N. 1. (34) Snyderman, S. E., L. E. Holt, Jr., P. M. Norton and E. Roitman. 1968. Effect of high and low intakes of individual amino acids on the plasma aminogram. Pages 19-31 in Protein nutrition and free amino acid patterns..1. H. Leathern, ed. Rutgers University Press, New Brunswick, N.J. (35) Welch, 1. G,, M. Claney, and G. W. VanderNoot. 1969. Net energy value of alfalfa and orehardgrass hays at varying stages of maturity. |. Anhn. Sci. 29:263. (36) Weston, R. H., and ]. P. Hogan. 1971. The digestion of pasture plants by sheep. V. Studies with subterranean and berseem clovers. Aust. J. Agar. Res. 22:139. (37) Wohlt, J. E., C. 1. Sniffen, and W. H. Hoover. 1973. Measurement of protein. solubility in common feedstuffs. J. Dairy Sci. 56:1052.

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