Supplemental yeast culture alters the plasma amino acid profiles of nursling and weanling horses

Equine Nutrition and Physiology Society REFEREEDPAPERSFROMTHE11THSYMPOSIUM

SUPPLEMENTALYEASTCULTUREALTERSTHE PLASMA AMINOACID PROFILESOF NURSLINGAND WEANLINGHORSES M. J. Glade, BS, PhD 1and M. D. Sist, BS, DV1VP

SUMMARY

The ability of a dietary supplement of dried live yeast culture to influence the growth rates, feed conversion efficiency and plasma amino acid profiles of young horses was tested. 10-week old nursling foals fed a creep feed of corn, oats and pelleted alfalfa, made available at 1% bodyweight daily, were also given 10 g yeast culture per day, mixed with granulated sugar and fed by hand. The nurslings remained with their dams and were allowed to continue to nurse freely throughout the 7 weeks of the experiment. In a second, preliminary trial, 6-9 month old weanlings were fed a ration consisting of crimped oats, alfalfa hay, a pelleted concentrate/roughage mix and cracked corn, at 2% of bodyweight daily, with or without supplemental yeast culture (20 g/day). Jugular venous blood samples were drawn from supplemented and unsupplemented animals during each trial after 0, 2, 4, 6, 8, 22 and 36 days, and from the nurslings again at 50 days. Significant increases in the plasma concentrations of arginine, glutamine, lysine, methionine and valine and significant decreases in ammonia, hydroxyproline and 3methylhistidine were observed in the supplemented nurslings after 6-22 days. Compared to an unsupplemented weanling fed the same diet, 4 supplemented weanlings exhibited significant increases in plasma arginine, glutamine, glycine, isoleucine, leucine, methionine and valine concentrations, while ammonia, hydroxyprolineand 3-methlylAuthors' addresses: ~Departmentof Pharmacology, Northwestern University, 303 East Chicago Avenue, Chicago, Illinois 60611. 21629 Meech, Williamston, Michigan 48895. Acknowledgements: Generous support for these studies was provided by AIItech Biotachnology, Lexington, KY. Portions of these data have been presented at the 100th Irish Veterinary Congress, Dublin, September 1988, and at the Tenth Equine Nutrition and Physiology Symposium, Stillwater, Oklahoma, May 1989.

Volume 10, Number 5, 1990

histidine concentrations decreased. Rates of gain were significantly increased in both groups of supplemented foals, and feed conversion efficiencyby weanlings improved 8% with supplementation. These findings suggest that supplemental yeast culture may influence amino acid balances and nitrogen metabolism in young horses, resulting in enhanced growth.

INTRODUCTION

The growth-limiting and growth-permitting roles of dietary protein are explicitly recognized in the utilization of total nitrogen content as the dominant criterion representing a ration's overall quality and value. However, the digestibilities of the nitrogen sources it provides are as important as the feed's total protein content. Even more important than the digestibility of a ration's nitrogen is the ability of the young horse to incorporate the absorbed nitrogen-containing compounds into body tissues. The efficiency of nitrogen retention will depend in part on the balance (relative proportions) of circulating amino acids being delivered to growing tissues. In the young horse, blood amino acid balance is fairly reflective of feed amino acid composition1 and will conceptually be more conducive to tissue growth as the biological value of feed protein increases. Commercially available dried yeast cultures have improved the biological value of the feed of yearling horses, by decreasing urinary nitrogen losses z3and encouraging the release of microbial amino acids into the digestive tract where they are available for absorption, a,4 Such beneficial effects may require a mature intestinal microfloral ecosystem. Younger horses lack fully-developed microflora, and may therefore be unable to respond as 369

•

Equ,no Nutr,t,onanO

h,s,o,oo, Soo,et,

RE ER EOP.PERS ROMT.E.T.SY.OS,

Table 1. Amino acid composition of experimental diets fed to nurslings and weanlings. Unsupplemented Feed Animo a c i d

Nurslings

W e a l i n g s Yeast Culture

--g/kg feed dry matter--

alanine arginine aspartic acid cysteine glutamic acid glycine histidine isoleucine leucine lysine methionine phenylalanine proline serine threonine tryptophan b/rosine valine amino acids crude protein

7.53 7.23 5.03 1.93 6.38 6.68 2.70' 6.43 11.28 5.50 2.10 6.61 9.03 5.85 5.56 2.03 5.26 7.20

104.33 138.60

7.78 8.26 5.25 2.14 6.73 7.07 3.06 6.67 10.78 6.33 1.95 6.89 9.27 5.83 5.60 2.14 5.47 7.58

108.80 143.30

g/kg dry matter 31.4 37.8 19.9 8.4 25.2 29.4 16.6 33.5 53.4 43.0 12.6 31.4 35.7 35.9 33.5 8.4 25.0 37.8

519.1 612.5

dramatically to yeast culture supplementation. More effective utilization of feed nutrients should be reflected in accelerated rates of gain and improved feed conversion efficiency. In addition, an improvement in circulating amino acid profiles is implicit in an improvement in feed biological value and nitrogen retention efficiency. The possibility that yeast culture supplementation could affect growth rates, feed conversion efficiency and plasma amino acid concentrations was tested in two groups of young horses, creep-fed nurslings with their dams and weanlings.

MATERIALS AND METHODS Nurslings

A group of eight Thoroughbred and Standardbred foals being raised on the same breeding farm participated in this study. The foals were all born within.a three-week period and remained with their dams and continued to nurse throughout the study. No creep feed was provided until each nursling reached 6 weeks of age, when it began receiving 370

creep feed in an individual creep arrangement inaccessible to its dam or other horses. Creep feed consisted of corn (25%), oats (25%), and pelleted alfalfa (50%), and was provided daily in amounts equal to 1% of each foal's estimatedbodyweight;bodyweightswere estimated weekly with a heart girth tape. Uneaten feed (averaging less than 3% of the original amount provided per foal per day) was removed and weighed daily. As the nurslings reached 10 weeks of age, they were alternately assigned to 2 treatment groups. Both groups continued to receive the same creep feed as before. In addition, one group received 10 g of a commercially-availabledried live yeast culture preparation (YEA-SACC. Alltech Biotechnology, Nicholasville, KY) mixed with granulated sugar and fed by hand once daily for 7 weeks. The palatability and acceptability of the supplement were high; all 4 foals were consistently consuming all of the supplement within 3 days. No subsequent refusals were noted and the level of animal cooperation remained high. The amino acid compositions of the creep feed and of the yeast culture supplement were determined by HPLC following hydrolysis in 8 N methanesulfonic acid at 155 C for 48 hours. 5 The amino acid composition of the experimental diets are given in Table 1. Ten g of yeast culture provided 5.2 g of additional amino acids and 6.1 g of additional crude protein per day. Total creep feed crude protein intake by nurslings was increased by about 0.6% of total creep feed dry matter intake. Consequently, crude protein, total amino acid and individual amino acid intakes of supplemented nurslings tended to be slightly greater than those of unsupplemented nurslings (Table 2). However, multivariate analysis of variance utilizing Wilks' criterion6 revealed no overall effects of supplementation on the amino acid intakes of the 2 groups of foals. The increases in daily crude protein intakes by supplemented nurslings averaged only 3.2 g/100 kg bodyweight/day, and the increases in lysine intakes averaged only 0.25 g/100 kg/day. None of the differences in individual amino acid intakes were significant (p>.05), when compared by analyses of variance for repeated measures. 6 Jugular venous blood samples were drawn from all foals in the early afternoon on days 0 (one day before any yeast culture was fed), 2, 4, 6, 8, 22, 36 and 50. Plasma was separated and deproteinized, and its free amino acid, ammo_n.i~ hydroxyproline and 3-methylhistidine contents determined by HPLC. The plasma amino acid concentrations of both groups over the 51 days of the experiment were EQUINE VETERINARY SCIENCE

Equine Nutrition and Physiology Society REFEREEDPAPERSFROMTHE11THSYMPOSIUM

Table 2 Mean dry matter, crude protein and amino acid intakes of nurslings fed creep feeds with or without supplemental yeast culture (YC).

Nutrient

YC

10

11

Weeks of aglea

12

13

15

16

17

pSEM a

g/lO0 kg bodyweight/day dry matter

891 885

crude protein

123.5 126.1

:i:~:i:i:i:i:!:

il

893 896

896 897

910 910

123.8

124.2 127.0

126.1 128.1

::::::::::::::::::::::::::::

~::::~~

127.2

93.17 96.10

93.48 95.94

94.94 97.58

6.72 6.90

6.75 6.89

6.85

6.46 6.67

6.48 6.65

6.58 6.74

4.49 4.61

4.51 4.59

4.58 4.65

1.72 1.77

1.73 1.77

1.81

5.70 5.84

5.72 5.83

5.81 5.90

5.97 6.13

5.99 6.12

6.08 6.20

2.41 2.51

2.42 2.51

2.46 2.53

5.74 5.93

5.76 5.92

5.85 5.99

10.28

10.07 10.38

10.11 10.35

10.26 10.48

lysine

4.90 5.21

4.91 5.16

4.93 5.14

5.01 5.20

methionine

1.87 1.93

1.88

1.88 1.94

1.91 1.97

amino acids

92.93 95.31 +:.:.:.:+

alanine

6.71 6.83

arginine

6.44 6.62

aspartic acid

4.48 4.56

cysteine

1.72 1.75

glutamic acid

5.68 5.78

glycine

5.95 6.07

histidine

2.41 2.49

isoleucine

5.73 5.88

leucine

i

~::#~

:::::::::::::::::::::::

~iiiiiiiiiiiil

:iN ::-~ i i~i~Niiiiiiii -::~N

10.05

phenylalanine

5.89 6.02

proline

8.04 8.19

sefine

5.21 5.40

1.95

608 6.o

iii"

,92

~i~:!ili~ili 6.o7 iiiiiiii 8 . .

6.97

1.79

6.02 6.14

8.25 ..............~.~~ii::iiil 8.25

8.22 8.35

5.22 ~ i ~i~ ii~ii~i 5.24 5.43

~!~ i::::iiiiiii

5.32 5.48 5.06 5.20

5.41

4.95 5.12

497

iiiiiiiiiiiii

498

5.16

~i~ iiiiiiiii

5.15

1.81 1.84

1.86

1.86

162

1.85 1.88

tymsine

4.69 4.80

4.70 iiiiiiiiiiiiiiiiiiiii~i~ii!iiii! 4.71 4.83 4.84 iiii::iiiiiii::!::iiii~i~::iii::iii i:~:i:~:~:~:~:i:i:i:i:

4.79 4.89

valine

6.41 6.59

6.43

6.55

thmonine tryptophan

181

iii iiiiiiiiii

~::~i!;ii;iii :i:i:i:i:i:i:i:i::.

)-~:i:~:!:i: ~:.:~:.:-:::::

6.65 ii~::i::iiiiiii::iii iE~......,iiiiiiiil

6.45 6.63

6.71

aStandard error of a mean, pooled experiment-wise. Volume 10, Number 5, 1990

871

~ ~ ~" '

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EquineNutritionandPhysiology Society ~ REFEREEDPAPERSFROMTHE 11TH SYMPOSIUM

analyzed by multivariate analyses of variance utilizing Wilks' criterion, 6 and the plasma concentrations of each amino acid on each sampling day were analyzed by analyses of variance for repeated measures. 8 Weanllngs Five Quarterhorse weanlings (6-9 months old) participated in a preliminary pilot study. These animals were housed individually within the same barn and were fed individually twice daily. Their diet consisted of crimped oats (50%), second cutting alfalfa (40%), pelleted mix (7.5%) and cracked corn (2.5%),and was provided at a daily rate of 2% of bodyweight. Feed analyses were identical to those carried out during the nursling experiment. After all of the animals had been on this diet for at least a month, four of the animals were randomly selected to receive a dietary supplement of 10 g yeast culture with each feeding, for 36 days. Crude protein intakes by the supplementedweanlings averaged 0.3-1.1 g/100 kg/day more than that of.the unsupplemented weanling (Table 3). However, the results of multivariate and univariate analyses of variance indicated that amino acid intakes were not significantly greater. Jugular venous blood samples were drawn in midmorning on days 0, 2, 4, 6, 8, 22 and 36. Plasma was analyzed as described above. In order to make useful comparisons between the unsupplemented and supplemented weanlings, it was assumed that the pooled standard errors determined for the data from the supplemented weanlings were also applicable to the data obtained from the single unsupplementedweanling. Univariate analyzes of variance for repeated measures experiments were then applied to the plasma amino acid and ammonia dam 6

RESULTS Nurslings The unsupplemented nurslings averaged 105.3 + 9.2 kg (SEM) at 10 weeks of age and 153.5 + 10.1 kg at 17 weeks of age, with an average rate of gain of 0.98 + 0.04 kg/ day. The supplemented nurslings averaged 118.0 + 5.0 kg at 10 weeks of age and 173.3 + 6.4 kg at 17 weeks of age. Their average rate of gain (1.13 + 0.04 kg/day) was significantly greater (p<.05) than was that of the unsupplemented nurslings. Heights at the withers at 10 weeks of age averaged 112.8 + 2.1 cm for the unsupplemented and 113.6 + 1.9 cm for the supplemented nurslings and were not significantly 372

different. At 17 weeks, withers heightsaveraged 123.6 + 2.8 and 127.0 + 1.9 cm for the unsupplemented and supplemented nurslings, respecti,bely. The supplemented nurslings grew an additional 2.6 cm on average during the 7 weeks of the experiment (p<.05). There were no differencesamong the 2 groups of foals in any of their individual plasma amino acid concentrations on day 0 (Table 4). However, their plasma amino acid profiles were significantly affected by subsequent yeast culture supplementation, as indicatedby multivariate analyses of variance. This overall effect became apparent between days 6 and 8, and remained through at least day 36. The plasma amino acid concentrations of several individual amino acids were significantly affected by yeast culture supplementation (Table 4). These effects were not concurrent, but appeared at various times after the beginning of supplementation. After 4 days of supplementation, plasma isoleucineconcentrationswere 38 % greater (13<.01) in the supplemented nurslings than in the unsupplemented horses at the time of sampling. Although isoleucineconcentrations remained higher in these foals through the remainder of the study, the differences were not statistically significant after 4 days. Similarly, plasma leucine concentrations tended to be relatively greater in the supplemented animals, beginning on the fourth day of supplementation, but because of high interanimal variability in leucine concentrations, the difference between groups were not statistically significant. In contrast, plasma arginine and valine concentrations were elevated 50-130% in the supplemented foals (compared to the unsupplementedfoals), beginning on day 6 and continuing through day 50. Plasma methionine concentrations were 38% greater (p<.05) in the supplemented horses on day 8 of supplementation, and the magnitude of the differenceincreasedthrough day 50 (to 82%; p<.O1). Plasma lysine concentrations were significantly increased by supplementation on days 8-36, but were no longer elevated on day 50. Plasma glutamic acid and glutamine concentrations also tended to be higher in the supplemented foals after day 6, although only the increases in glutamine concentrations on days 22, 36 and 50 were statistically significant. Plasma ammonia concentrations were significantly decreased within 2 days of supplementation. This decrease persisted through the 50th day of supplementation and averaged 23-54%. Plasma hydroxyproline and 3-methylhistidine concentrations also progressively decreased during the 50 days of supplementation. In contrast to most other amino acids, plasma histidine concentrations tended EQUINEVETERINARYSCIENCE

Equine Nutrition and Physiology Society REFEREEDPAPERSFROMTHE11THSYMPOSIUM -~a:~3~ ~ Table 3. Mean dry matter, crude protein and amino acid intakes of weanlings fed diets with or without supplemental yeast culture (YC).

Nutrient

YC

1

2

Week of Experiment 3 4

5

6

pSEM=

g/lO0 kg bodyweighUday dry matter

+

crude protein

266.5 267.3

266.5 267.4

+

202.4 203.1

202.4 203.2

alanine

!iii!iiiiiii~i~iiiiiiiiiiiiilj ii 267.0 266.5

iiiiiiiiiiii~i~i:iiiii!ii!iiiii!i

i~iiiiiiiiii~i;iiiiiii!ii!iii ii~iiiiiliiiii~i~ ili~,iiii;,

202.4 202.9 14.47 14.51

14.47 14.51

14.47 14.51

+

15.36 15.42

15.36 15.42

9.77 9.79

9.77 9.79

+

3.98 3.99

3.98 3.99

+

12.52 12.54

12.52 12.55

iiii!iii!!!!;~i~!iiiiiiiiiii iiiiiiiiiiiiiiii~i~iiiiiiiiiii! i i i i i i i i i i i i i~i~i i i i !iiiiiiiiiiiiiii~i~iiiiii iiiiiiiiiiil!ii~i~!iiiiiii ~i::i!i i;:ii;i i i;i i i~:~i~i i i i i i l

÷

13.15 13.18

13.15 13.19

::iii::i~i~ii:~ii::::~i::~i~t:~iii;iI!:=i

+

5.69 5.72

5.69 5.72

12.41 12.46

12,41 12.46

12.41 12.45

+

cysteine

-

glutamic acid

histidine isoleucine ÷

~i~iiiiiiiiiiiii~i~i;;i;iiiii!i!ii!

15.36 15.40 9.77 9.78

i i i i i i iiiiiiiii~!iiiiiiiii il i i~i i i iiiiii~iiiiiiiiiii i i i i!i i iiiiiiiii~ii!i~ i iiiiitilili~M~ii~ii!

3.98 3.99 12.52 12.55

~iiiiiiiii i i ilji~iiii~iiiiiiiiiiii 13.15 13.18 i i i i i i i i i i~i i l~i i i i l iiiiiii!ii!iiiii~i~iiiiiiiil 5.69 ::ii#i i i::!::i::::!i::ii#:Si~Si::ii i i i i 5.71 ili i l i~i i i~l i i i ili~i ~i ~i i i~i~

iiiiiiii iii

+

20.05 20.13

20.05 20.14

20.05 20.10

+

11.77 11.86

11.77 11.86

11.77 11.85

iZi i i i i i i i i i i i i i i~i i i i i i i i i i i lili i i~i i i i i ili~i i i i i i

leucine lysine

i i i i i i i i i i i i i i i~i i i i i i

÷

3.63 3.65

3.63 3.65

3.63 3.65

+

12.82 12.86

12.82 12.87

12.82 12.84

÷

17,24 17.28

17.24 17,29

17.24 17.26

÷

10.84 10.91

10,84 10.91

10.84 10.89

÷

10.42 10.48

10.42 10.48

10.42 10.47

÷

3.98 3.99

3.98 4.00

3.98 3.98

÷

10.17 10.21

10.17 10.22

10.17 10.19

iiiiiiiiiiiii;ili~iii@iiiiiii

,4-

14.10 14.15

14.10 14.16

14.10 14.14

iiiiiiii

methionine

phenylalanine praline serine ~hr~nine

tryptophan l~/rosine

1860 1854

+

aspartic acid

gly~ne

1860 1856

+

amino acids

aNinine

1860 1855

-

valine aStandarderrorof a mean

Volume 10, Number 5, 1990

i i i i i i i i i i i i i i i i i i i i i i i i i i i~i l iiiii~iiii~ii~iiiiiiiiiiiiii~iiiiiiiiii

iiii!iiii!!!i!iiii~!iiiiiii!!ii~ii!!!iiii! i ji i i i i i i i i i i i ~i~i i i i i

i 373

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EquineNutritionand PhysiologySociety .OM

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to be decreased in the supplemented foals beginning on day 6. Despite the various changes in individual amino acid concentrations, total plasma free amino acid concentrations were not significantly affected by yeast culture supplementation.

Weanlings The supplemented weanlings gained 28.75 + 3.10 kg during the 5 weeks of supplementation, compared to 23.00 kg gained by the unsupplemented weanling: The feed-togain ratios of the supplemented weanlings averaged 7.11 + 0.50, an average of 8% less than that of the unsupplemented weanling. Changes in withers heights during the 5 weeks were unaffected by yeast culture supplementation (data not shown). The plasma amino acid profiles of the 5 weanlings were not different at the beginning of supplementation but were significantly affected by 1 week or more of supplementation (Table 5). After 36 days of supplementation, plasma arginine, glutamine, glycine, isoleucine, leucine, methionine and valine concentrations were significantly increased in the supplemented weanlings, compared to the unsupplemented weanling. In contmsL plasma ammonia, hydroxyproline and 3-methylhistidine concentrations were decreased. Unlike the nurslings, the weanlings did not exhibit any transient changes in plasma amino acid concentrations during the 36 days of observation and sampling.

DISCUSSION These results confirm that very young horses are capable of responding to oral supplementationwith a fermentation-enhancing preparation of dried live yeast culture.

Rates of gain and linear growth were both stimulated by the supplementation of the creep feed of nursling foals. These effects were statistically significant and the data in Table 4 reinforce the conclusion that it is justifiable to assume that the 2 groups of mare-foal units were comparable when the diet assignments were made. However, because lactation was not examined it is not possible to estimate quantitatively the true extent of the supplement's impact on nursling growth. Acceleration of tissue growth requires an increased migration of tissue substrates from the digestive tract to target sites via the circulation. Altered migration might be exhibited by changes in the relative steady-state plasma concentrations of tissue substrates. Although interanimal variation might in some cases be quite large, it is reasonable to assume that plasma samples drawn between nursing bouts by freely-nursing and creep-feed nibbling young foals would on average be fairly reflective of steady-state conditions, given the rapidity of digestion, absorption and passage of oral suspensions within the equine alimentary system. 7 The plasma concentrations of several amino acids were affected in the supplemented nurslings. Perhaps most importantly, plasma lysine concentrations were significantly elevated after a week of supplementation. Lysine is widely recognized to be the most growth-limiting amino acid for foals, 8 and increased plasma lysine concentrations have been associated with significantly accelerated growth during the first 2 months of equine life.6 In addition, the plasma concentrations of two indirect indicators of tissue catabolism were reduced in the nurslings during yeast culture supplementation. Plasma hydroxyproline concentrations gradually decreased during supplementation and were significantly reduced after 36 days. Changes in circulating hydroxyproline concentra-

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Table 4. Mean plasma amino acid concentrations in nursling foals fed diets with or without supplemental yeast culture (YC).

Amino acid

YC

0

2

Days

4

6

8

22

36

50

p S E M=

rag/all alanine

+

arginine + aspo~tic acid +

asparagine +

.653 .647

.742 .716

i::i:i:i1!:i:!!:i~i i:i:::i

1.390 1.425

1.205c 2.184

1.228c 2.569

.670 .648

.630 .653

.660 .628

.065 .067

.067 .067

.068 .059

o69 iiii~iiii .o~ i::II!iiiN~!~iii

llliiiiiii~ ii!tlI

iiiiiiliiii i!llil iiii!liliiii~ !iiii!! iiiiliii iili

705 i l i i ~

716 iiiiiiliNii 1.544

.58o

iiii!ii

.163 .189

.225 .148

.156 .155

.,= .189

+

.775 ~iI!!i:.iiii.Ii1-21£i~i~i~i~i!i~ .790

,815 .873

.750 .875

., .580

.601 .667

.625 .680

.595b .914

.786

1.490 !!Iiiii.-Ii.'(.!~1i!I!!ii 1.038

1.270 1.250

1,740 .995

.603

7.570 6,260

10.395¢ 6.190

7.645 6.823

9.860 7.973

+

1.739 1.524

1.262 1.584

.777 .957

.543 .705

1.986 1.889

2.013 2.079

1.416

+

2.157

1.559 2.321

glutamic acid glutamine 4-

glycine +

histidine +

isoleucine

leucine

iiiiiilNiiiiii~iiiiiiiil

!iiii!ii

!il!i iii::!iiili ilii IiINiil

iiiilIii~iii!

1.378

1.409

1.475¢

1.322

4-

1.528

1.486

1.861

1.248

1.340 1339

1287

4-

1.318

1.420b 1.983

1.118¢ 2.033

1.222

lysine methionine

+

1.705

1.223 1.593

.719 .990

.671 .937

+

1.648 1.538

1.605 1.682

1.633

1.681

1.508 1.665

+

2.095 2.695

2.230 1.538

1.365 1.728

1.470 1.970

4-

1.910 1.653

1.860 1.643

1.775 1.478

1.840 1.610

.658 .506

.540 .581

.620 .583

.635

4-

.480 .577

.444 .546

.542 .603

.336 .394

phenylalanine

serine thr~nine

.545 .634

I!iili~i iiii

+

~steine

praline

~iii::i~ iiiiii

-

tryptophan tyrosine

i~ii!ii!i

iiN !iiiNi

,553

continued next page

Volume 10, Number 5, 1990

375

Equine Nutrition and Physiology Society

Table 4 continued

iiii!:~:~:i:iii:~:i:i: ii!i~iiiiiii!iiiii!ii~ii

valine

total free amino acids

iiiili:iliiiiiiiiiiii

i~iiiiiiiill i i~ii~i::i::i::iii~3.284

+

ili;iiiiiiiii~~ONiii!i!iii 25.480

+

. 28o iiii!iiii!i i i!i

.298o i!iiiii!i!iiii!iiiii! .285°

• ~98

iiiiiiiiiiiiiiiiiii~#i

.212

•765

iiiiiiii~ i ~

.767

ammonia hydroxyproline

i ;i i i l

+

3-methyihistidine

iiiiii!iiiiiiiiilTi i

+

+

3.928

iiiiii!iiiiii!~iiiiiiiiiiiii

3.2o2

!iiiii!i i i!iiiiii 2 .828 iiiiiii i iiii!iii2 . 25

.855

iiiN!i!iiiiii

;~iiiiiiiii;~ii~::iii::i::::i

ii!iiiiii~iiiiiiiil

•1 4 0

•130

i3i3iiii.:.:'i;i;~i;::.'l~::iiiii;i

25.716

.649

iiiiiiiii~i~i::i::i::i::iiii25.620

iiiiii!iiiiiiiiiiiii~i~iiiiiiiiiill .~ z

iiiiiiii~iiilii .786b

iiiiii:i:iiii iiiii!iiiiii !iii!iiii iiiiiiii::ii~ii~ii!iiii!iiiiia.,,~8 iiiilNi i!ii}iiiiiii iii#iiiN!iiiiiiiiill .252° iiii !i!i iiiiiiiiiiiiiiiiiii::Niiiiiiiiiiiiiii

.146

~i i i:i.--,"~..'..~i:;!i ~!i!~i

.781c

::i::ii!iiiiii::::::iiii~iiiiiiiii::i!i .615

.120 ?ii?iiNiii~;t! " ::?!i?!? .133 b i!~!~;~ii~i,:':.ii~~:::~!~iii:. ! ::-! ,115 ili;i::ii~iiii::i::i::i~::~;~liiiiiiiii::iii;.088 i~i~i::i~i~i~i::ii::i~i~::ii~

.146c .079

aStandard error of a mean, pooled experiment-wise. bFor a given number of days, means for supplemented and unsupplemented horses are significantly different, p<.05. ¢For a given number of days, means for supplemented and unsupplemented horses are significantlydifferent, p<.01.

tions generally are assumed to reflect changes in skeletal remodeling, with decreases in plasma hydroxyproline concentrations reflecting a reducedrelease of this metabolite of proline from its major tissue store, collagen. ~°Because the primary source of collagen turnover in the growing animal is bone, ~1 changes in plasma hydroxyproline concentrations are considered to parallel changes in bone resorption, with decreases in both suggesting a net increase in bone formation. ~a,~a Should decreased circulating hydroxyprolineconcentrations indicate a decelerationof bone resorption in young horses, the decreases exhibited by yeast culture supplementednurslings might be taken to reflectenhanced skeletal development. Similarly, changes in plasma 3-methylhistidine concentrations are crude indicators of the relative rate of muscle myofibrillar protein degradation. ~4 The decreases observed in the supplemented nurslings are consistent with more efficientgrowth of protein stores. It is well established that protein degradation and synthesis by skeletal muscle in tissue culture are sensitive to the concentrations of the branched chain amino acids in the medium. 15,16Leucine is a particularly potent inhibitor of myocytic protein degradation 15.~7and stimulator of intracellular protein synthesis.~5 In the supplemented nurslings, plasma valine were significantly elevated and leucine and isolencine concentrations tended to be higher throughout supplementation. Together with the observed decreases in plasma 3-methylhistidine concentrations, these data suggest that yeast culture supplementation may have induced shifts in net muscle protein metabolism toward increased net muscle gain. Supplementedfoals also exhibited sustained increases in plasma concentrations ofmethionine,an important source

376

of the sulphur and methyl groups required for muscle and connective tissue synthesis. ~s The effects of yeast culture supplementation on the circulating concentrations of the branched chain amino acids also suggest an overall improvement in muscle energetics. Studies in other species have revealed an association between elevated circulating branched-chain amino acid concentrations and a shift in the energetics of muscle toward decreased intracellular protein degradation and alanine production. ~9.~o2~These shifts are accompaniedby decreased hepatic gluconeogenesis from alanine, decreasing hepatic production of ammonia,z2Interestingly, plasma ammonia concentrations were significantly reduced during supplementation, although because of the complexity of ammonia metabolism the biological meaning of this observation is not clear. However, in addition to potentially altering the balance of muscle protein turnover, yeast culture supplementation may help to "protect" young horses from ammonia sensitivity, a3 Many of these effects on amino acid metabolism and availability were also observed in the supplemented weanlings. In addition, the efficiency of feed conversion to body mass was also improved by supplementation, lending further support to the hypothesis that yeast culture supplementation may function as a natural biological growth promotam in immature horses. However, caution must be exercised when attempting to interpret the weanling data; they were obtained from a very preliminary examination in which only 1 unsupplemented animal served as a basis for comparison with the supplemented animals. Nonetheless, the data that were obtained did tend to be consistent with the data obtained from the nursling experiment.

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Table 5. Mean plasma amino acid concentrations in weanlings fed diets with or without supplemental yeast culture (YC).

Days Amino acid

YC

2

4

.777 ;;iiiiiiiiii~i;;;iii;iiii~i!;il

alanine

6 .777

....,..-..:-:-:-::::::i:i:i:~:i:

11~5 iiiiiiiiiiiiiii~iii~iil

arginine

1.125 iiiiiiii~iiiiiiiiiii~iii~ilil

+

iiiiiiiiiiiiii i

36

pSEMa

.768 .832

.018

1.103c 1.803

.056

.553 .610

.039

.079 .068

.008

.179 .163

.038

.630 iiiii::::::i::iii!iiiiiii}}i)~]~ii;::!

6o8

i i i i!i i i i i :~i~~ii~i~i~ iiiiiiiiiiiiiiiiiiiiiiN!iiii .600 .633

+

.068 .067

.065 .065

+

.155 .156

.189 .191

.615 .625

.640 .633

.640 .688

.035

+

.477 .528

.491 .493

.508c .719

.026

+

.444 .496

.456 .508

.465c .669

.035

+

+

6.925 6.700

6.850

+

.725 .720

-

1.033

1.130

+

1.095

1.293

1.263

.960 .903

1.005 .910

1.015 .970

.059

+

1,025 1.048

1,045 1.053

1.045c 1.345

.043

+

+

.946 .935

.833 .855

.753 .820

.062

asparagine cysteine

glutamic acid glutamine

glycine his~dine

isoleucine

lysine

methionine

7.025 .768c 1.005

i i i i i i iiiii! i i i i i !i i i i i i i !iiii i

7.300 6.675

.590

.695c .913

.060

1.o55b

.068

i i i i ;i;;ii i i•;!i

1.588 1.583

1.628 1.643

1.655 1.618

.070

+

-

iiiiiiiiiiiiiiiiiiiiili

1.868 1.728

1.945

2.018 1.920

.091

1.928

proline serene

1.100 1.205

22

+

aspartic acid

phenylalanine

!~iiiii!~iiiiiii~iiiiii

.816 iiiiiii,,i,~,,;~,,iiiN.~i~;~; .829 i!i i i i i i ;i i i i i i ~i~::ii i ili

+

leucine

8

:::i:i:: :i: :::"': :~

+

}~iiii~:ii~i~;ili}i

÷

iii!::i::::iiii!i!is~!iiii.~

threonine trytophan +

tyrosine +

v~ine

iiiiiiiiiiiiiiiiiii iliiiiiiiiiiiiiiiiii!i iili!iiiiiiii! i ii

1.275 1.435

1.198

1.245 1.3OO

.108

1.403

.600

.618 .538

.638 .560

.037

.563 iiiiii!ii!!!~:iiiii~!ii!~i~i~!i!

478 iiiiiiiiiiiiiiiiiii!iiiii ii

.480

.468 .494

.018

.491 2.410b 2.660

2.430c 2.815

.085

.625 iliiii!ii;iiiiiii !Niiiii

2.630 i::;!{~i::i::iil;!i::;::~!~}i}i

continued next page

Volume 10, Number 5, 1990

377

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Table 5 continued

i i i i~i~i i i i i i~i i i~i i i~i i~i~i i i i~i i~i i

to lfree aminoacids

iiiiiiiiiiiiiiiiiiiiiiii!ii~iiiiiii!iiiiiiiiiiiiiiiiii

iiiiiii!iiiii!iiiii!iii!iiiii ! 26.504 iiii!i!i!i!i!i!i!iiiiiiiiiiii i 26.716 +

iiiiiiiiiiiiiiiiiiiiiiii~i~iiiii

26.591 !iiiiiiiiiiiiiiii~#~iiiiiiiiiiii27.306

i i i i i i i i i~i i i i i~i::ii i i i i i i!~ili::i::i

!iii!iiii~i..".:iiiiiiii!!i!i~ii~ii

26.887 iiiii::ii!iiiiii~i~iiiii~ii!!i~i26.166

!!i!iiiiiiiiiiii~i~iiiiii !iiiiiiiiiiiiiiiii~i~iiiiii

.353

.268 c ;iii~;i!i iii3!i!iiiii~6~iii

.012

........,,,.......................................

hydroxyproline

+

;;;;;;ii;iii;iiii!iiiiiii!iii!ii!ii~iiii!i

.265 !iiii;i;;;iiiiiii;ii;ii!;iii~iiiiii;;~;ii .213

-

iiiiiiiiiiiiililiiiiiiiiiiililili~l iii

.768 iiiiliiiiiiiliiiiiiiiiiiii!~!!!!!iiiii!i

+

::::::::::::::::::::::::::::::::::::::::::: :::::::::::::::::::::::::::::::::::::::::::::::::::::::

.7~0 iiiiiiii~iiiiiililililiiii~i!i!i!!!!~!i .715

3-methyl-

histidine

+

iiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiii~iii iiiiiiii;;;ii;;iii;iii;iiiiiiiiiiii~i~iiiiil

.:+:+:.:.:.:.:.:,:,:,:.:.:.:+:+:.:.:.:+:+:

.181 ii;iii;ii;ii!iil;i;iii~i~;!i iiiiiiiiiiiiiii!!i!!!!iiiiii !iiii !i!iii! ~iiiiiiiiiiiiiiiiiiiiii.~ii iii.iiiiiiiiiiiiiiiii ::::::::::::::::::::::::::::::::::::::::::::::::::::

::::::::::::::::::::::::::::::::::::::::::::::::::::::

.778b iiiiiiiiiiiiiiiiiiiiii!i!i!i~ili .069 :+:+:.:.:.:+:+:.:+:.:+:.:.:.:.:.:+:+

iiiiiiiiiiiiiii!ii!ii!!~!~!!!!!!!!ii!!i!i!i!i!iii!iii:

.138 iiiiiiiiiiiiiiiiiiiiiiiiii;i~i~!ii!i;iiiiii .123 .128 i;iiiiiiiii?;;!!ii!i!!i!!ii~iiiiiiiiiiii

................................

.718

.113

i i!i!il!iili!iiiiiiiiii i

.....,.....,..,.......................

.575 !i!iiiiiiiiiiiiiii!i!:!i!i!! iiiil;

ii!ii!i i

.128o iiiiiiiiiiiiiiiiii i i i

.013

:::::::::::::::::::::::::::::::::::::::::::::::::::.075 iiiiiiii!!iiiiiiiiiiiiiiiiii iiiiii

aStandarderror of a mean, pooled experiment-wise. bFor a given numberof days,meansfor supplementedand unsuppiementedweanlingsare different, p<.05. CFora given numberof days, means for supplementedand unsupplementedweanlingsare different, p<.01.

The magnitude and timing of the changes in plasma amino acid concentrations indicate that, rather than merely directly supplying additional nutrients to the blood, yeast culture supplementation acted indirectly to alter equine amino acid metabolism.There is evidence to suggest that the effects on amino acid physiology were secondary to an enhancement of fiber fermenting capabilities and interactions with the urea recycling system of the large intestine. 4 Dietary supplementation with yeast cultures has been reported to facilitate the fermentation of dietary fiber, T M with a subsequent increase in the apparent digestibility of feed protein via an increased rate of urea recycling into usable amino acids4 that can be absorbed along the equine hindgut, a4,asThese processes have resulted in a doubling of the percentage of digested nitrogen that was retained within the body tissues of yeast culture supplemented yearling horses, a suggesting that increasing the effectiveness of urea recycling does enhance the biological value of ingested protein. This conclusion is consistent with the elevations in plasma amino acid concentrations observed in yeast culture supplemented nurslings and weanlings. In addition, increased fiber fermentability also increases the energy available for additional tissue growth (in effect, increasing feed DE content), a,a,4 These results confirm that even the very young equine digestive system is responsive to the stimulation of fermentation by yeast culture supplements. These observations begin to define the role played by yeast culture, and potentiaUy by other probiotics, in increasing the efficiency of utilization of dietary nutrients and enhancing muscle and bone growth.

378

REFERENCES 1. Comerford PM, Edwards RL, Hudson LW, Wardlaw FB: Supplemental lysine and methionine for the equine. S. C. Agric Tech Bull 1073,1978. 2. Godbee R: Effect of yeast culture on apparent digestibility and nitrogen balance in horses. Clemson Univ Res Bull, 1983. 3. Glade MJ, Bieski LM: Enhanced nitrogen retention in yearling horses supplemented with yeast culture. J Anita Sci 62:1635,1986. 4. Glade MJ, Sist MD: Dietary yeast culture supplementation enhances urea recycling in the equine large intestine. Nutr Rep Intern 37:11,1988. 5.Simpson RJ, Neuberger MR, Liu T-Y: Complete amino acid analysis of proteins from a single hydrolysate. J Biol Chem 251:1936,1976. 6. Cochran WG, Cox GM: ExperimentalDesigns. John Wiley and Sons (NY), 1957. 7. Glade MJ, Luba NK: Serum tri-iodothyronine and thyroxine concentrations in weanling horses fed carbohydrate by direct gastric infusion. Amer J Vet Res 48:578,1987. 8. Ott EA, Asquith RL, Feaster JP: Lysine supplementation of diets for yearling horses. JAnim Sci 53:1496,1981. 9. Glade MJ, Luba NK: Benefits to foals of feeding soybean meal to lactating broodmares. Proc Equine Nutr Physiol Symp p.593,1987. 10. Klein L, Albertsen K, Curtiss PH Jr: Urinary hydroxyproline in hyperparathyroidism: a study of three cases with and without bone lesions. Metabolism 11:1023, 1962. 11. Perez-Tamayo R: Pathology of collagen degradation. Amer J Pathol 92:509,1978. 12. Lemmo EA, Evans JL: Serum hydroxyproline as an index

for calcium nutriture,an interactanton tissue zinc and copper in growing, pregnant and lactating rats. In: Trace Substance in Emdronmental Hea/th-XI/I, ed. Hemphill DD, University of Missouri (Columbia), p.461,1979. 13. EvansJL, VerdarisJN, Kim CW: Serum hydroxyprolineas an indexofcatcium nutrition and the mobilizationof skeletalreserves. IV Intl Symp on Ruminant Physiol Handbook, p.61,1974.

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14. Young VR, Munro HN: N-methylhistidine(3-methylhistidine) and muscle protein turnover: an overview. Fed Proc 37:2291,1978. 15. Skjaerlund DM, Mulvaney DR, Mars RH, Schroeder AL, Stachiw MA, Bergen WG, Merkel RA: Measurement of protein turnover in skeletal muscle strips. J Anim Sci 66:687,1986. 16. U JB, Jefferson LS: Influenceof amino acid availabilityon protein tumover in peffused skeletalmuscle. Biochem BiophysActa 544:351,1978. 17. SchneiblePA, Airhart J, Low RB: Differentialcompartmentation of leucine for oxidation and for protein synthesis in cultured skeletal muscle. J Bio/Chem 256:4688,1981. t8. MaynardLA, Locsli JK, Hintz HF, Warner RG: AnimalNutr 7th Ed. McGraw-HillBook Co.(NY) 1975. 19. Rennie I~J, Edwards RHT, HallidayD: Protein metabolism during exercise. In: ifitrogen Metabolism in Man, ed. Waterlow JC, Stephen JML, Applied Science Publishers(London), 1981.

20. Tischler ME, Goldberg AL: Amino acid degradation and effect of leucine on pyruvate oxidation in rat atrial muscle. Amer J Physiol 238:E480,1980. 21. Chau BHL, Siehl DL, Morgan HE: A role for leucine in regulation of protein turnover in working rat hearts. Amer J Physiol 239:E510,1980. 22. Felig P, Wahren J: Amino acid metabolismin exercising man. J C/in/nvest 50:2703,1971. 23. Hintz HF, Lowe JE, Clifford AJ, Visek WJ: Ammonia intoxicationresultingfromurea ingestionby ponies. JAmer VetMed Assn 157:963,1970. 24. Slade LM, Bishop R, Morris JG, Robinson DW: Digestion and absorptionof l~N-labelledmicrobialprotein in the large intestine of the horse. Brit VetJ 127:11,1971. 25. Lew JP, Baker JP: Amino acid absorption by the equine intestine. Proc Equine Nutr Physic~ Symp, unpaged addendum, 1983.

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