SORBITOL CLEARANCE AND ITS EFFECTS ON FEEDLOT

SORBITOL CLEARANCE AND ITS EFFECTS ON FEEDLOT PERFORMANCE

AND CARCASS CHARACTERISTICS OF STEERS

by

DENNIS WELDON BOYLES, JR., B.S., M.S.

A DISSERTATION

IN

ANIMAL SCIENCE

Submitted to the Graduate Faculty of Texas Tech University in

Partial Fulfillment of the Requirements for

the Degree of

DOCTOR OF PHILOSOPHY

Approved

December, 1993

M

ACKNOWLEDGMENTS C7^^ ""

J) 1 would like to express my gratitude to Dr. C. Reed

Richardson for his support during my graduate studies. He

has helped instill in me a deep appreciation of the

metabolic processes involved in nutrition.

I am grateful to have had distinguished men serve on my

committee. A sincere thank you goes to Drs. R. L. Preston,

R. C. Albin, D. Oberleas and N. A. Cole for their support

and encouragement.

I would like to thank Dr. G. S. Cameron (Texas Tech

Health Sciences Center, Department of Dermatology) for his

vital service to me while laboring through this research.

His guidance, assistance, friendship and the use of his

laboratory are deeply appreciated. I would also like to

thank the staff at the Burnett Center, Victor M. Montalvo-

Lugo and fellow graduate students for their assistance and

friendship.

The support of family and close friends is acknowledged

and appreciated. But the support and years of sacrifice

unselfishly supplied by my wife Janie and our children,

Colton, Caylen and Kaycie, have made the completion of this

research possible.

11

TABLE OF CONTENTS

ACKNOWLEDGMENTS ii

LIST OF TABLES v

LIST OF FIGURES vii

LIST OF ABBREVIATIONS ix

CHAPTERS

I. LITERATURE REVIEW 1

Introduction 1

Animal Performance 1

Metabolism of Sorbitol 2

Properties of Sorbitol 4

Rumen Fermentation of Sorbitol 5

Monosaccharide-Amino Acid Interactions 6 Summary 7

II. EFFECTS OF SORBITOL ON FEEDLOT PERFORMANCE AND CARCASS CHARACTERISTICS OF STEERS FED A STEAM-FLAKED GRAIN SORGHUM BASED DIET 9

Abstract 9

Introduction 10

Materials and Methods 10

Results and Discussion 12

Implications 14

III. EFFECTS OF PROTEIN SOURCE AND SORBITOL SUPPLEMENTION ON PERFORMANCE OF INCOMING FEEDLOT STEERS 28

Abstract 28

Introduction 28



Implications. 31

• • •

111

IV. EFFECTS OF SORBITOL ON SODIUM DEPENDENT AND SODIUM INDEPENDENT GLYCINE AND LEUCINE UPTAKE BY CULTURED BOVINE KIDNEY CELLS 38

Abstract 38

Introduction 38


Results and Discussion 4 3

Implications 44

V. CLEARANCE OF INTRAVENOUSLY

ADMINISTERED SORBITOL IN STEERS 49

Abstract 49

Introduction 50



Implications 57

VI. INTEGRATED SUMMARY 66

LITERATURE CITED 69

APPENDIX

A. CELL SUBCULTURE TECHNIQUE 75

B. CELL FREEZING TECHNIQUE 77

C. EXPERIMENTAL PROCEDURE FOR TREATMENT OF

CELLS IN COSTAR CLUSTER WELLS 78

D. PROCEDURE FOR PROTEIN DETERMINATION 80

E. CONSTRUCTION OF EXPERIMENTAL WASH TRAYS 81

F. CONSTRUCTION OF EXPERIMENTAL UPTAKE TREATMENT TRAYS 82

G. FRUCTOSE DETERMINATION IN BOVINE PLASMA 83

IV

LIST OF TABLES

2.1 Composition of initial backgrounding diet 15

2.2 Composition of second backgrounding diet 16

2. 3 Composition of third backgrounding diet 17

2.4 Composition and analysis of final basal diet 18

2.5 One hundred-nineteen day feedlot performance 19

2.6 Initial twenty-eight day feedlot performance 20

2 .7 Initial fifty-six day feedlot performance 21

2.8 Initial eighty-four day feedlot performance 22

2.9 One hundred-twelve day feedlot performance 23

2 .10 Carcass characteristics 24

2.11 Carcass quality grades by treatment 25

3.1 Composition of basal diet 33

3.2 Treatment means for twenty-eight day feedlot performance 34

4.1. Effect of sorbitol on sodium dependent and independent glycine uptake by bovine kidney cells 45

4.2. Effect of sorbitol on sodium dependent and independent leucine uptake by bovine kidney cells 46

5.1. Composition and analysis of basal diet 58

5.2. Mean plasma glucose of steers receiving an intrajugular infusion of either glucose, sucrose, sorbitol or propionate in 50% solutions at 2.2 g kg"^ metabolic weight or equal volume of saline 59

5.3. Mean plasma sorbitol of steers receiving an intrajugular infusion of sorbitol in a 50% solution at 2.2 g kg"^ metabolic weight or equal volume of saline 60

5.4. Mean plasma fructose of steers receiving an intrajugular infusion of sorbitol in a 50% solution at 2.2 g kg"l metabolic weight or equal volume of saline 61

VI

LIST OF FIGURES

2.1. Gain efficiency of steers fed for 119 d 26

2.2. Average daily gain of steers fed for 119 d 27

3.1 Dry matter intake of steers fed either low ruminally degradable protein (LD Protein) mixture or readily ruminally degradable protein (RD Protein) supplement 35

3.2. Average daily gain of steers fed either low ruminally degradable protein (LD Protein) mixture or readily ruminally degradable protein (RD Protein) supplement 3 6

3.3. Gain efficiency of steers fed either low ruminally degradable protein (LD Protein) mixture or readily ruminally degradable protein (RD Protein) supplement 37

4.1. Effects of sorbitol on glycine uptake by cultured bovine kidney cells in the presence of sodium (glycine NA) or in the absence of sodium (glycine WO) 47

4.2. Effects of sorbitol on leucine uptake by-cultured bovine kidney cells in the presence of sodium (leucine NA) or in the absence of sodium (leucine WO) 48

5.1. Plasma glucose levels of steers receiving an intrajugular infusion of either glucose, sucrose, sorbitol or propionate in 50% solutions at 2.2 g kg~^ metabolic weight or equal volume of saline 62

5.2. Plasma glucose levels of steers receiving an intrajugular infusion of either sucrose, sorbitol or propionate in 50% solutions at 2.2 g kg~^ metabolic weight or equal volume of saline 63

5.3. Plasma sorbitol levels of steers receiving an intrajugular infusion of sorbitol in a 50% solution at 2.2 g kg"^ metabolic weight or equal volume of saline 64

Vll

5.4. Plasma fructose levels of steers receiving an intrajugular infusion of sorbitol in a 50% solution at 2.2 g kg~^ metabolic weight or equal volume of saline 65

Vlll

LIST OF ABBREVIATIONS

ADG Average daily gain

BRSV Bovine respiratory syncytical virus

BVD Bovine viral diarrhea

Ca Calcium

cm Centimeters

CP Crude protein

CV Coefficient of variation

d Day(s)

DMI Dry matter intake

DM Dry matter

F:G Feed to gain ratio

g Grams

G:F Gain efficiency (g of gain / kg of feed)

h Hour(s)

HCWT Hot carcass weight

IBR Infectious bovine rhinotracheitis

K Potassium

kg Kilogram(s)

Meal Megacalories

w^5 Metabolic weight (body weight in kg*^5)

NEg Net energy for gain

NEm Net energy for maintenance

P Phosphorus when referring to diet composition

P Probability when referring to statistics

PI3 Parainfluenza

REA Ribeye area

SEM Standard error of the mean

VFA Volatile fatty acid(s)

IX

CHAPTER I

REVIEW OF LITERATURE

Introduction

Sorbitol, a natural six-carbon polyalcohol, is found in

various human foods such as cherries, plums, pears and

apples (Merck, 1989). It is also used as a sweetener. This

compound is regarded as safe for human consumption and is

classified as GRAS (generally recognized as safe) by the

Food and Drug Administration.

Supplementation of sorbitol has been shown to improve

performance of veal calves (Thivend, 1982; Bauchart et al.,

1985). Sorbitol addition to a corn silage-based diet has

also been shown to improve feedlot performance of steers and

bulls. Fontenot and Huchette (1993) reported improvements

in feed efficiency by steers without improved daily gains

after Geay et al. (1992) described improvements in both feed

efficiency and daily gains by bulls. Since cattle feeders

use feed additives and implants to improve animal

performance, sorbitol could be viewed as a natural, safe

compound that could be used as a livestock feed additive

provided it increases profits.

Animal Performance

When orally administered to ruminants, sorbitol has

been shown to improve feed efficiency in calves from birth

to three weeks of age (Daniels et al., 1981) and between 8

and 16 weeks of age in veal calves (Thivend, 1982; Bauchart

et al., 1985). Thivend et al. (1984) have reported an

increase in biliary organic matter flow of young calves when

consuming sorbitol. Improvements in feed efficiency when

supplementing sorbitol to ruminants used for beef production

has also been documented in two reports. Geay et al. (1992)

reported that sorbitol supplied at a concentration of 50 g

d~^ improved ADG and feed efficiency by finishing bulls.

Recent feedyard performance trials conducted by Fontenot and

Huchette (1993) have shown that feeding 20 to 4 0 g of

sorbitol d~^ to finishing steers improves feed efficiency

while not affecting ADG, in two of three studies, with no

marked effect on carcass characteristics. Supplementary

studies conducted by Fontenot and Huchette (1993) indicate

that the improvement in feed conversion by ruminants is not

due to differences in rate of passage or digestibility.

Bauchart et al. (1985) reported a 7% improvement in feed

efficiency from sorbitol supplementation to young veal

calves with no differences in digestibility.

Recent work conducted in France (Fostier, 1992) has

shown that large doses (one kg per animal) of sorbitol given

orally via the drinking water reduced the incidence of dark

cutting carcasses from Holstein cows (n = 2000) by 30%.

Also increased muscle glycogen concentrations in the

longissimus muscle of bulls was reported following sorbitol

consumption. Sorbitol's involvement in muscle glycogen

metabolism has been shown earlier by Johnston and Deuel

(1943) using rats.

Metabolism of Sorbitol

The metabolism of sorbitol is closely related to that

of fructose and glucose (Bye, 1969). Sorbitol dehydrogenase

and aldose reductase are the two enzymes involved in the

metabolism of sorbitol. Large amounts of sorbitol

dehydrogenase are found in hepatocytes and in very low

concentrations in other tissues (Blakley, 1951; Bye, 1969;

Seeberg et al., 1955). Aldose reductase is largely located

in the kidney (Stribling et al., 1989) and plays a minor

role in sorbitol metabolism (Bye, 1969). Early research

indicated that sorbitol dehydrogenase is highly specific for

D-sorbitol, but more recent reports suggest that other

polyols are metabolized by sorbitol dehydrogenase (Malone

and Lowitt, 1992). Sorbitol dehydrogenase catalyses the

following reactions in the presence of NAD or NADH

(Bergmeyer, 1974):

D-Sorbitol 4-> D-Fructose

L-Iditol <-> L-Sorbose

Ribitol 4-> D-Ribulose

Xylitol <^ D-Xylulose Allitol <- D-Allulose

L-Gala-D-glucoheptide <-> L-Galaheptulose

D-Altro-D-glucoheptide <- D-Altroheptulose.

This enzymatic activity involving other polyols could

possibly be related to the improvement in cattle feedlot

performance when cattle consume carrots that contain

mannitol (Richardson, 1993).

Evidence that fructose is the primary intermedate of

hepatic sorbitol oxidation leading to glucose production was

reported by Embden and Greesbach (1914). The enzymatic

oxidation of sorbitol to fructose leading to glucose

production was confirmed by Blakley (1951) in rats.

Sorbitol was rapidly oxidized by rat liver slices;

therefore, Blakley (1951) concluded that in liver slices the

oxidation of sorbitol to fructose with subsequent conversion

of fructose to glucose proceeded much more rapidly than

other hexose oxidation. Chemical reactions involved include

two pathways (Lehninger, 1982). The major pathway in

muscles and the kidney is as follows: sorbitol <-> fructose

<-> fructose-6-phosphate <-> glucose-6-phosphate <-> glucose.

In the liver, the pathway includes three more intermediates

according to the following: sorbitol <-> fructose <->

fructose-1-phosphate <-> glyceraldehyde-3-phosphate <->

fructose-l,6-diphosphate <-> fructose-6-phosphate <-> glucose-

6-phosphate <r> glucose.

Properties of Sorbitol

Sorbitol is (1) rapidly removed from the blood (Stetter

and Stetter, 1951; Hoshi, 1963). Molino et al. (1987) have

described the hepatic clearance of sorbitol to be such that

sorbitol is essentially metabolized and completely extracted

by the hepatocytes. Gianpaolo (1991) states that the

hepatic uptake of sorbitol is by passive transport and the

transport mechanism is unsaturable in the absence of any

substrate competition. Bye (1969) found that sorbitol was

rapidly removed from the blood of 16 humans during and

following intravenous infusion of sorbitol with only 3% of

administered sorbitol escaping via the kidneys. Sorbitol is

presently used in medicine as a compound for the non

invasive test in evaluating liver plasma flow because of its

biologic and kinetic (first-order) features. For a compound

to be used in the non-invasive test in evaluating liver

plasma flow, it must be a substance which is essentially

metabolized by the hepatocytes and completely extracted at

any passage through the liver (Waldstein and Arcilla, 1958;

Winkler et al., 1979; Winkler et al., 1973). The hepatic

clearance of sorbitol largely exceeds liver blood flow

(Molino et al., 1987; Bass and Winkler, 1980). The liver is

a highly vascular organ. In sheep the fraction of cardiac

output received by the liver is 30-40%. The liver contains

an enormous capillary bed which aids in the special effect

that the hepatic parenchyma cells have on substances

entering the organ (Smith and Hamlin, 1977). The amount of

cardiac output flowing through the liver and the enormous

vascular system it possesses sheds light on the rapid

clearance of sorbitol. (2) Sorbitol has been reported to

exhibit protein-sparing properties (Kaufmann, 1929; Griem

and Lang, 1960). Bye (1969) reported that sorbitol was used

as the main source of calories when human patients were fed

intravenously for three months without alteration in

nitrogen balance. Adcock and Gray (1956) concluded that

sorbitol does provide calories to both normal and diabetic

humans based on data collected after an oral dose of 35

grams of ^^c sorbitol. (3) Sorbitol possesses antiketogenic

properties that have been reported to be superior to

fructose. Early work reported by Edson (1936) showed that

sorbitol was more effective than fructose or glucose in

suppressing spontaneous ketogenesis in liver slices obtained

from starved rats.

A vitamin-sparing action of sorbitol has also been

reported. Morgan and Yudkin (1957) have shown that sorbitol

produces a change in the intestinal flora of rats so that

thiamin is synthesized and is available to the host. The

tissues of rats surviving on thiamin-free diets plus

sorbitol contained more thiamin than those rats on thiamin-

free diets when glucose was supplemented. Thus thiamin was

made available to the tissues, though not from the diet.

Supplementary studies conducted by Morgan and Yudkin (1957)

suggested that the administration of sorbitol appears to

make the rat independent of dietary sources not only of

thiamin but also of the other B-complex vitamins. This

reported action of sorbitol's involvement in thiamin

metabolism in rats if functional in ruminants could possibly

play a preventive role when cattle suffer from

polioencephalomalacia (Blood and Henderson, 1974).

Rumen Fermentation of Sorbitol

Little information concerning rumen fermentation of

sorbitol exists. No differences in VFA molar proportions 6

h after finishing bulls were fed 0 or 50 g of sorbitol d~l

were reported by Geay et al. (1992). However, they did find

an increase (P < .001) in propionic acid production in vitro

with a decrease in butyric and isovaleric acids along with a

reduction (P < .001) in the acetic:propionic ratio.

Improvements in feed efficiency elicited by sorbitol appears

to decline with time. The decline has occurred after 56 to

70 d when ruminants were fed 20 to 50 g of sorbitol animal"^

d"l without altering rate of passage or digestibility

(Fontenot and Huchette, 1993; Geay et al., 1992,

respectively). Geay et al. (1992) postulated that the

decline in the beneficial effect of sorbitol in improving

performance by rumanints could be attributed to two factors:

a decreasing growth potential of of the host and an

increasing capacity of ruminal microorganisms to metabolize

sorbitol.

Monosaccharide-Amino Acid Interaction

Monosaccharides such as glucose, galactose and fructose

derived from dietary sources have been reported to alter

amino acid transport (Reiser and Hallfrisch, 1987). Glucose

and galactose have generally inhibited the transport of

neutral amino acids, while fructose, a derivative of

sorbitol, has been documented to increase neutral amino acid

transport. In vitro, cellular uptake of leucine,

isoleucine, valine, phenylalanine, tryptophan and histidine

were increased 24 to 57% by the presence of fructose in the

incubation media (Reiser and Christiansen, 1971; Reiser et

al., 1975; Reiser and Hallfrisch, 1977). In vivo, fructose

has also stimulated neutral amino acid transport by intact

intestine (Reiser and Christiansen, 1969; Alvarado, 1968).

The effectiveness of fructose in stimulating leucine

transport appears to exist whether fructose occupies

intracellular or extracellular space (Reiser and Hallfrisch,

1987). They also have stated that the component of leucine

transport stimulated by intracellular fructose is dependent

on metabolic energy and the presence of a sodium gradient.

Sorbitol is a monosaccharide closely related to fructose.

Sorbitol and fructose are enzymaticaly interconverted by

sorbitol dehydrogenase (SDH; EC 1.1.1.14). Fructose is

reduced according to the following reaction:

Sorbitol + NAD"*" <- SDH _ Fructose + NADH + H"*".

Since sorbitol is a monosaccharide closely related to

fructose, and fructose has been reported to affect amino

acid transport, the effect of sorbitol on amino acid

transport should be examined.

Summary

In summary, data suggest that sorbitol is oxidized more

rapidly than other hexoses (Blakley, 1951), it is

antiketogenic (Edson, 1936) and its primary role in

metabolism pertains to energy utilization. Whether this

effect results from conversion of sorbitol into energy

intermediates or due to gastrointestinal tract changes that

promote better energy utilization such as the vitamin-

sparing action or due to some protein-sparing effect or to

all of the above is at present unknown.

Improvements in feed efficiency and sometimes ADG when

feeding sorbitol to ruminants has been reported by several

researchers. One could hypothesize that sorbitol improves

performance of cattle by providing added substrate used in

gluconeogenesis because sorbitol is quickly converted to

fructose and then to glucose. Since ruminants depend upon

gluconeogenic precursors (i.e., propionate and amino acids)

for their glucose needs, the gluconeogenic property of

sorbitol could possibly provide ruminants an additional

gluconeogenic precursor. Additionally, monosaccharides

closely related to sorbitol differentially affect amino acid

transport. It has not been determined if sorbitol affects

amino acid transport.

The mechanism and(or) mechanisms involved, associated

with sorbitol, in improving performance of ruminants is

presently unknown. Therefore, it was the intent of the

following research to determine the effects of sorbitol

supplementation to a steam-flaked grain sorghum-based diet

fed to steers; to determine the effects of sorbitol on amino

acid uptake by cultured bovine kidney cells; and to

determine sorbitol clearance in steers.

8

CHAPTER II

EFFECTS OF SORBITOL ON FEEDLOT PERFORMANCE

AND CARCASS CHARACTERISTICS OF

STEERS FED A STEAM-FLAKED

GRAIN SORGHUM-BASED DIET

Abstract

A feedlot experiment with growing/finishing steers was

conducted to determine the effects of supplementing a steam-

flaked grain sorghum-based diet with sorbitol on voluntary

feed intake, rate of gain, feed efficiency and carcass

characteristics. One hundred twelve crossbred steers

(Angus/Hereford; average initial shrunk weight = 337.3 ±

17.4 kg) were randomly assigned to treatment and fed a

steam-flaked grain sorghum-based diet with or without

sorbitol. Treatments were: A - control (basal diet); B -

basal diet plus 30 g sorbitol steer"^ d"^; C - basal diet

plus sorbitol at a variable rate (20 g sorbitol steer"^ d~^

first 28 d, 30 g second 28 d, then 40 g until termination of

the study); D - basal diet for first 69 d then addition of

30 g of sorbitol steer"^ d~^ until termination of the study.

Supplementation of sorbitol did not statistically improve

steer feedlot performance (P > .05) over the 119 d feeding

period. However, throughout the feeding period,

supplementing steers with 30 g of sorbitol d"^ showed a 3.4%

numerical increase in ADG and a 4.0% numerical improvement

in feed efficiency over steers receiving no sorbitol.

Steers receiving sorbitol only for the final 50 d had lower

dressing percent (P < .002) than other steers. Furthermore,

the carcass lean color tended (P = .16) to be altered by a

treatment effect . Steers receiving 30 g of sorbitol d"^

appeared to exhibit a more youthful bright cherry red color

of the carcass lean (P = .03). In summary, sorbitol

supplementation to a steam-flaked grain sorghum-based diet

did not improve feedlot performance of steers in this study.

However, steers fed 30 g of sorbitol d~l for the last 50 d

did exhibit (P < .002) lower dressing percentages.

Furthermore, steers receiving 30 g of sorbitol d~^ appeared

to exhibit (P = .03) a more youthful bright cherry red color

of carcass lean.

Introduction

Sorbitol, a natural six-carbon alcohol found in many

fresh fruits, has been reported to improve feed conversion

in calves (Daniels et al., 1981; Thivend, 1982; Bauchart et

al., 1985) and in finishing bulls (Geay et al., 1991). Two

of three feedlot experiments reported by Fontenot and

Huchette (1993) have shown improvements in feed efficiency

by finishing steers when sorbitol was supplemented to a corn

silage-based diet alone or in combination with monensin.

Thus, the objectives of this study were to determine the

effect of sorbitol supplementation to a steam-flaked grain

sorghum-based diet with monensin on feedlot performance and

carcass characteristics of growing/finishing steers.

Materials and Methods

One hundred and twelve crossbred steers (Angus/

Hereford; average initial shrunk weight = 337.3 ±17.4 kg)

were randomly assigned to treatment and fed a steam-flaked

grain sorghum-based diet with or without sorbitol.

Treatments were: A - control (basal diet); B - basal diet

plus 30 g sorbitol^ steer"^ d~^; C - basal diet plus

sorbitol at a variable rate (20 g sorbitol steer"! d~! first

1 Neosorb^Sorbitol supplied by Roquette Corporation, Gurnee, IL 60031-2392.

10

28 d, 30 g second 28 d, then 40 g until termination of the

study); D - basal diet for first 69 d then addition of 30 g

of sorbitol steer"! d"! until termination of the study.

Upon arrival at the research facility steers were all

weighed, ear tagged, and vaccinated against BVD, BRSV, IBR,

PI32, and Clostridium perfringens Types C and D^. Steers

were gradually placed on the final basal diet using three

diets over 28 d by decreasing roughage source and increasing

the amount of steam-flaked grain sorghum. Each

backgrounding diet (Tables 2.1, 2.2 and 2.3) was fed for at

least five d. The steers were implanted with Synovex-S^ at

the start of the experiment and at d 56. Steers were

weighed initially and at 28 d intervals through 112 d.

Steers reached market weight by 112 d and remained on

treatment an additional 7 d until slaughter was scheduled.

At the beginning and end (119 d) of the feeding period two

consecutive weights were taken to obtain full and shrunk

weights. Shrunk weights were obtained after 24 h without

feed and overnight without water. Additionally, steers fed

sorbitol starting on d 70 (treatment D) were weighed on d 69

before the morning feeding.

All diets were formulated to meet or exceed NRC (1984)

recommended requirements for CP, Ca, P and K. The final

basal diet is shown in Table 2.4. Sorbitol was added (via a

premix using ground grain sorghum as the carrier) to the

diets at 1% of diet DM. The amount of sorbitol premix was

2 Horizon IV. Bovine Rhinotracheitis-Virus Diarrhea-Parainfluenza3-Respiratory Syncytial Virus Vaccine, Modified Live and Killed Virus, Diamond Scientific, Company, Des Moines, lA.

3 Clostridium perfringes Types C & D, Coopers Animal Health, Inc., Kansas City, KS 66103.

4 Syntex Animal Health, 4800 Westown Parkway, W. Des Moines, lA 40265.

11

adjusted daily if needed to ensure proper sorbitol

consumption. Steers were maintained on treatment for 119 d.

Two steers were removed from the experiment; one steer on

treatment D died from bloat 22 d after initiation of the

study, and the other steer was removed from treatment B due

to hoof injury on d 84. Carcass data were obtained at a

commercial beef packing plant by trained university meat

science personnel. Livers were scored using the Elanco

abscessed liver classification system^.

There were four replications (pens) of seven steers

pen"! on each of the four treatments. Pens of steers served

as the experimental units in a completely randomized design.

The model included treatment and data were analyzed by

analysis of variance using the General Linear Model

procedure of SAS (1990). Treatment means were separated by

Tukey's Studentized Range test.

Results and Discussion

Supplementation of sorbitol over the 119 d feeding

period tended to alter gain efficiency (g of gain kg"! of

feed; P = .09; Table 2.5) of steers while DMI (P = .59) and

ADG (P = .19) were not affected. Numerical improvements

were exhibited by steers fed 30 g of sorbitol d"! (treatment

B) throughout the feeding period in G:F (P > .05; Figure

2.1) and ADG (P > .05; Figure 2.2) compared to steers fed no

sorbitol (treatment A) (175 vs. 168 [4.0 %] and 1.46 vs.

1.51 [3.4 % ] ; G:F and ADG; A and B, respectively; Table

2.5). Data summarized throughout the feeding period (Tables

2.6, 2.7, 2.8 and 2.9) indicate the numerical improvements

in steer performance from supplementing 30 g of sorbitol.

Steers receiving sorbitol at the variable rate (treatment C)

5 Liver Abscess Technical Information. Elanco Products Company, Cattle Products Marketing, Lilly Corporate Center, Indianapolis, IN 46285.

12

had lower ADG (P < .05) and G:F (P < .05) for the initial 28

d when fed 20 g of sorbitol d"! (Table 2.6), and thereafter

their performance appeared to be adversely affected (P >

.05), as exhibited in Tables 2.6, 2.7, 2.8 and 2.9.

Carcass data are shown in Table 2.10. Steers receiving

sorbitol only after 69 d (treatment D) had lower dressing

percentages (P < .002) than steers receiving the three other

diets. Dressing percent (hot carcass weight/live weight x

100) pertains to the carcass yield. It is a function of

gastrointestinal fill and carcass fat; therefore, fatter

cattle will usually dress higher (Romans et al., 1985). The

steers in this study were managed in the same manner, so it

was unlikely that fill was a factor. Carcass data such as

ribeye area and fat measurements did not show strong

differences that would help explain why the steers fed 30 g

of sorbitol d"! for last 50 d had lower dressing

percentages.

Quality grades were similar (P = .60). When observing

the quality grades (Choice, Select or Standard) assigned to

carcasses within each treatment as shown Table 2.11, it

appears that steers fed sorbitol had fewer carcasses that

graded Choice. Overall, the percentage of carcasses that

graded choice was 19% which is undesirable. Based on

carcass value, the target set for carcasses grading Choice

is at least 60%. The genotype and weight of these steers

would indicate a higher percentage of carcasses grading

Choice. The reason for the low percent of carcasses grading

Choice in this experiment is unclear. An overall treatment

effect tended (P = .16) to alter the color of carcass lean

tisssue. Steers supplemented sorbitol at a constant rate of

30 g d"! (treatment B) appeared to exhibit (P = .03)

carcasses with a more youthful bright cherry red color of

lean tissue.

13

As shown in Table 2.10 sorbitol tended to affect the

severity (P = .17) and the incidence of liver abscesses

(P = .11) of steers. An important note to realize when

considering the liver abscess data in this experiment is the

absence of antibiotic formulated into the diets as requested

by the funding agency.

In conclusion, the addition of sorbitol to steam-flaked

grain sorghum-based diets when fed to growing/finishing

steers in this experiment did not improve feedlot

performance of the steers. This is in contrast to results

reported by Fontenot and Huchette (1993) when finishing

steers were fed a corn silage-based diet supplemented with

sorbitol. However, steers fed 30 g of sorbitol d"! for the

last 50 d did exhibit (P < .002) a lower dressing

percentage. Also steers receiving 30 g of sorbitol d~! for

119 d exhibited (P = .03) a more youthful bright cherry red

color of carcass lean.

Implications

Supplementing sorbitol to steam-flaked grain sorghum-

based diets in this experiment did not improve feedlot

performance of steers. This is in contrast to data reported

by Fontenot and Huchette (1993) and Geay et al. (1992).

They reported that feedlot performance was improved when

sorbitol was supplemented to high corn silage-based diets.

Carcass characteristics were not altered except for (1)

steers fed 30 g of sorbitol d"! for the last 50 d had lower

dressing percentages (P < .002), and (2) steers receiving 30

g of sorbitol d~! for 119 d appeared to exhibit (P = .03) a

more youthful bright cherry red color of carcass lean.

Except for these two carcass characteristics, carcass data

exhibited in this study support earlier work reported by

Fontenot and Huchette (1993) and Geay et al. (1992).

14

Table 2.1. Composition^ of initial backgrounding diet.

Item Percent

Corn silage 56.40

Cottonseed hulls 31.00

Cottonseed meal 7.11

Urea .16

Molasses, cane 2.50

Animal/vegetable fat 1.20

Calcium carbonate .36

Dicalcium phosphate .20

Sodium chloride .16

Trace mineral premix^ .17

Vitamin A premix^ .21

AS700 premix^ .53

^ As-fed basis.

b Contained (ppm) I 1,232, Mn 8,069, Zn 8,409, Cu 827, Co

51, Fe 4,056.

c Contained 660,000 lU vitamin A kg"!.

^ Contained 6.16 g aureomycin and sulfamethazine kg"!.

15

Table 2.2. Composition^ of second backgrounding diet.

Item Percent

Corn silage 40.00

Steam-flaked grain sorghum 20.60



Urea .25

Molasses, cane 3.30




Sodium chloride .11


Vitamin A premix^ .25

AS7 00 premix^ :_50

^ As-fed basis.


51, Fe 4,056.

c Contained 660,000 lU vitamin A kg"!.

^ Contained 6.16 g aureomycin and sulfamethazine kg"!.

16

Table 2.3. Composition^ of third backgrounding diet

Item Percent

Steam-flaked grain sorghum 62 . 05



Urea .56

Molasses, cane 3.85




Sodium chloride .21

Potassium chloride .09


Vitamin ADE premix^ .33

Rumens in premix^ .42

^ As-fed basis.

^ Contained (ppm) I 1 232, Mn 8 069, Zn 8 409, Cu 827, Co

51, Fe 4056.

^ Contained (lU/kg) Vitamin A acetate 634,480; Vitamin D 63,448; Vitamin E 1,813.

^ Contained monensin at 3,044.8 mg/kg.

17

Table 2.4. Composition^ and analysis of final basal diet.

Item Percent




Urea .37

Molasses, cane 3.27


Calcium carbonate 1.27

Sodium chloride .17



Rumensin premix^ .84

Control premix® .93

Sorbitol premix^ .00

Analysis^

DM, % 83.03

CP, % !3.03

.67

.32

.67

Ca, %

P, % K, % NEm, Meal/kg 3.78

NEg, Mcal/kg !'25 ^ As-fed basis. b Contained (ppm) I 1,232, Mn 8,069, Zn 8,409, Cu 827, Co 51, Fe 4056. c Contained (lU/kg) Vitamin A acetate 634,480; Vitamin D 63,448; Vitamin E 1,813. ^ Contained monensin at 3,044.8 mg/kg. e Contained 2.0% mineral oil and 98.0% ground grain sorghum f Contained 2.0 % mineral oil, 23.6% ground gram sorghum and 74.4% sorbitol. g Analyzed, except for NEm and NEg, which were calculated, on a DM basis except for DM.

18

Table 2.5. One hundred-nineteen day feedlot performance^

Treatment

A B C

Item

Variable Start 30

Control 30 g/d rate g on d 70 SEM^ P

Initial

wt^, kg

DMI, kg

ADG, kg

G:F^

F:Ge

Final

wt, kg

340.5

8.68

1.46

168

5.93

514.2

338.6

8.65

1.51

175

5.73

518.3

330.9

8.39

1.38

164

6.08

495.1

334.5

8.48

1.46

172

5.80

508.2

8.68

.174

.040

2.71

.094

16.15

.35

.59

.19

.09

.09

.19

^ 119 d performance using shrunk weights.

^ Standard error of the mean.

^ Average initial shrunk weight after one d off feed and

without water overnight.

^ Gain efficiency = g of gain kg"! of feed.

® Feed to gain ratio.

19

Table 2.6. Initial twenty-eight day feedlot performance^.

Item

DMI,

ADG,

G:F®

F:Gf

kg

kg

A

Control

8.09

2.39C

295C

3.39C

Tr€

B

30 g/d

7.94

2.35C

296C

3.37C

jatment

C

Variable

rate

7.62

1.95^

256^

3.91^

D

Start 30

g on d 70

7.79

2.I2C

272Cd

3.5lCd

SEM^

.159

.111

9.08

.115

P

.25

.04

.02

.02

^ 28 d performance summary.


C/d Means in same row with different superscript differ.

® Gain efficiency = g of gain kg"! of feed.

^ Feed to gain ratio.

20

Table 2.7. Initial fifty-six day feedlot performance^

Treatment

A B C D

Item

DMI,

ADG,

G:FC

F:G^

kg

kg

Control

8.50

1.97

232

4.32

30 g/d

8.33

1.97

236

4.23

Variable

rate

8.04

1.81

225

4.44

Start :

g on d

8.15

1.90

233

4.30

30

70 SEM^

.143

.049

4.77

.089

P

.16

.13

.45

.45





21

Table 2.8. Initial eighty-four day feedlot performance^

__ ^ ^ __ ^ Treatment

A

Item Control

B C D

Variable Start 30

30 g/d rate g on d 70 SEM

DMI,

ADG,

G : F C

F:G^

kg

kg

8 . 6 6

1 . 7 2

1 9 9

5 . 0 4

8 . 5 1

1 . 7 7

2 0 8

4 . 8 1

8 . 2 7

1 . 5 9

192

5 . 2 0

8 . 2 9

1 . 6 6

2 0 0

5 . 0 0

. 1 6 5

. 0 5 6

3 . 8 7

. 0 9 7

. 3 2

. 1 8

. 1 0

. 1 0





22

Table 2.9. One hundred-twelve day feedlot performance^

Treatment

A B C D

Item Control

Variable Start 30

30 g/d rate g on d 70 SEM

DMI,

ADG,

G : F C

F:G^

k g

k g

8 . 7 0

1 . 6 7

1 8 8

5 . 3 2

8 . 6 7

1 . 6 8

194

5 . 1 5

8 . 3 8

1 . 5 4

1 8 3

5 . 4 7

8 . 4 7

1 . 5 8

1 8 5

5 . 3 6

. 1 7 5

. 0 4 2

3 . 8 0

. 1 0 8

. 5 3

. 1 3

. 2 9

. 2 9





23

Table 2 . 1 0 . Carcass c h a r a c t e r i s t i c s .

Treatment B

Item Variable Start 30 g

Control 30 g/d ra te on d 70 SEM^

HCWT^*, k g

Dress ing % REA®, cm2

B a c k f a t ^ ,

cm

KPH, %

Y i e l d

graded

Marbling

score^

Quality

grade^

Lean

colorD

Liver

score^

318.4

62.1^

74.5

1.30

1.75

3.1

316.0

61.5C

76.5

1.24

1.69

2.9

307.2

61.9^

75.5

1.22

1.85

2.9

452.8 437.5 444.5

10.7

4.7

.70

10.3

5.3

.80

10.5

5.0

1.17

312.6

59.8«i

79.4

1.09

1.76

2.6

434.5

10.3

4.9

.64

4.91

.287

2.23

.102

.059

.199

11.7

.218

.165

.170

34.5 9.40

43

0004

49

85

36

44

,70

60

16

17

11

Liver abscess incidence!, %

39.3 38.6 66.7 ^ Standard error of the mean. ^ Hot carcass weight. C/d Means in same row with different superscript differ. ® Ribeye area in square, cm. f Backfat thickness, cm. g Calculated using USDA yield grade equation. h Marbling score; slight = 400; small = 500. i Quality grade; Choice"= 12; Select"*"=ll; Select"=10. J Lean color; light = 8; dark = 1. ^ Liver score using the Elanco abscess liver classification system. ! Liver abscess incidence by treatment; percent of steers with at least one liver abscess.

24

Item

Choice"

Select"*"

Select"

Standard"*"

A

Control

32.1

10.7

50.0

7.2

B

Treatment

30 g/d

15.4

15.4

57.7

11.5

C

Variable

rate

15.4

26.9

53.8

3.9

D

Start 30 g

on d 70

14.8

7.4

74.1

3.7

^ Percent of carcasses in each grade by treatment.

25

310 T

'S290 fSi ^ 2 7 0 o

^ 2 5 0 ••

1 2 3 0 ^ 2 1 0

^190

0 170

150 28 56 — \ —

84 Days

112 119

-•- Control -©-Variable

^ 30 g/d -^ 30 g/d last 50 d

Figure 2.1. Gain efficiency of steers fed either basal diet (Control), 30 g sorbitol steer"! d"! (30 g/d), variable rate (Variable) (20 g first 28 d. 30 g second 28 d, then 40 g of sorbitol steer"! (j-l until termination) , or 30 g sorbitol steer"! j-l only after 69 d (30 g/d last 50 d) (SEM = 2.71).

26

2.40 T

2.20 -

2.00 -^

01.80 -

^1.60 +

1.40 +

1.20 28 56

-•- Control -©-Variable

+ + 84 112 119

Days

^ 3 0 g/d -& 30 g/d last 50 d

Figure 2.2. Average daily gain of steers fed either basal diet (Control), 30 g sorbitol steer"! ^-1 (30 g/d), variable rate (Variable) (20 g first 28 d, 30 g second 28 d, then 40 g of sorbitol steer"! ^-1 until termination), or 30 g sorbitol steer"! d"! only after 69 d (30 g/d last 50 d) (SEM = .040).

27

CHAPTER III

EFFECTS OF PROTEIN SOURCE AND SORBITOL

SUPPLEMENTATION ON PERFORMANCE

OF INCOMING FEEDLOT

STEERS

Abstract

A 28 d experiment was conducted to evaluate the

effects of protein source and level of sorbitol

supplementation on performance of incoming crossbred steers

(262 ±21.7 kg; n = 260) using a randomized block design

with a 2 X 3 factorial arrangement of treatments. Protein

source was either a low ruminally degradable (LD) mixture or

a readily ruminally degradable (RD) protein supplement.

Sorbitol was fed at either 0, 30 or 60 g steer"! d"!.

Feedlot performance measures (DMI, ADG, gain efficiency,

G:F, g of gain kg"! feed) were not improved by feeding LD

protein or by sorbitol supplementation during- the 28 d

receiving period. However, there was a tendency (P = .18)

for an interaction between protein source and level of

sorbitol for gain efficiency.

Introduction

Sorbitol has been reported to improve feed conversion

in calves and finishing steers (Fontenot and Huchette, 1993)

and finishing bulls (Geay et al., 1992) when supplemented in

corn silage-based diets. Geay et al. (1992) fed sorbitol

(50 g d"!) plus low ruminally degradable protein to

finishing bulls and reported increased ADG (+ 18%) and feed

efficiency (+ 14%). However, when supplementing sorbitol to

a steam-flaked grain sorghum-based diet only numerical

improvements in feed efficiency and daily gains by finishing

steers have been reported (Boyles and Richardson, 1993).

28

Cattle arriving at feedlots are usually stressed

because of transport and from being without feed; thus these

animals are depleted of nutrients. This problem is

confounded by the low DMI of these cattle during the

receiving period. Thus, it is important to formulate

receiving diets properly to restore lost body nutrients.

Eck et al. (1988) suggested that incoming feedlot cattle

have a requirement for low ruminally degradable protein.

Therefore, because of the benefit of supplementing sorbitol

to veal calves, steers and bulls in conjunction with

evidence for improved 28 d performance of cattle fed a

source of low ruminally degradable protein, it seemed

fitting to conduct a 28 d receiving study to evaluate the

effects of protein source and level of sorbitol

supplementation on performance of incoming crossbred steers.


A 28 d study was conducted to evaluate the effects of

protein source and level of sorbitol supplementation on

performance of incoming crossbred steers! (262 ± 21.7 kg; n

= 260) using a randomized block design with a 2 x 3

factorial arrangement of treatments. Pen location within

the feedlot served as blocks. Protein source was either a

low ruminally degradable (LD) protein mixture (Preston and

Bartle, 1989; blood meal, 15%; corn gluten meal, 8%;

cottonseed meal, 22%; hydrolyzed feather meal, 33%; and meat

and bone meal, 22%) or a more readily ruminally degradable

(RD) source (soybean meal). Sorbitol was supplemented at 0,

30 or 60 g steer"! (j-l. Upon arrival at the Texas Tech

feedlot, steers were weighed, ear tagged, dewormed^ and

! Source: Oklahoma City Livestock Auction, OK.

2 ivomec-F, MSD-AGVET, Division of Merck and Company, Inc., Pathway, NJ 07065.

29

immunized against BVD, BRSV, IBR, PI33 and Clostridium

perfringens type C and D^. Steers were fed a steam-flaked

grain sorghum, cottonseed hull-based diet (Table 3.1) for 28

d. Sorbitol^ (granular form) was supplemented via a premix

with ground sorghum used as the carrier. Sorbitol premix

(85% sorbitol, 13% ground grain sorghum plus 2% mineral oil)

and a control premix (contained 98% ground grain sorghum

plus 2% mineral oil and no sorbitol) were adjusted daily if

needed based on pen feed intake to insure proper sorbitol

consumption.

There were five replications (pens) of eight or nine

steers pen"!, on each of the six treatments. One pen of

nine steers fed LD protein plus 30 g sorbitol d"! were

removed from analysis because the diet was inadvertently

changed to LD protein without sorbitol seven d after

initiation of the experiment. Pens of steers served as the

experimental units in a randomized block design with a 2 x 3

factorial arrangement of treatments. Pen location served as

the blocks. The model included blocks, protein source,

sorbitol level and protein source*sorbitol level

interaction. Data were analyzed by analysis of variance

using the GLM procedure of SAS (1990).


No interactions existed between protein source and

level of sorbitol for DMI (P = .75; Figure 3.1). There was

3 Horizon IV. Bovine Rhinotracheitis-Virus Diarrhea-Parainfluenza3-Respiratory Syncytial Virus Vaccine, Modified Live and Killed Virus. Diamond Scientific, Company, Des Moines, lA.

4 Clostridium perfringens Types C & D, Coopers Animal Health, Inc., Kansas City, KS 66103.

5 Neosorb^ Sorbitol supplied by Roquette Corporation, Gurnee, IL 60031-2392.

30

a tendency for an interaction between protein source and

level of sorbitol for ADG (P = .20; Figure 3.2) and gain

efficiency (P = .18; Figure 3.3). The low ruminally

degradable protein mixture tended to lower DMI, not affect

ADG and tended to improve G:F (Table 3.2; 8.0 vs. 8.21, P =

.20; 2.09 vs. 2.06, P = .69; 262 vs. 250, P = .21; DMI, ADG,

and G:F for LD vs. RD, respectively). Feedlot performance

of steers was not improved by sorbitol supplementation

(Table 3.2; 7.96, 8.30, 8.04, P = .27; 2.02, 2.16, 2.04, P =

.44; 254, 260, 255, P = .90; DMI, ADG and G:F, for 0, 30 and

60 g of sorbitol, respectively).

The overall means and corresponding coefficients of

variation (CV) for DMI, ADG and G:F were 8.11, CV = 5.58%;

2.01, CV = 11.16%; and 256, CV = 8.95%, respectively. The

average DMI of steers in this receiving study was 2.8% of

body weight (body weight = average of initial and 28 d

weights). Dry matter intake of this magnitude for incoming

steers is quite good. Therefore, a possible explanation for

the lack of statistical effect found in this experiment

could be associated with the DMI of these steers during the

28 d receiving period.

In conclusion, performance of steers during the 28 d

receiving period was not improved by feeding a low ruminally

degradable protein mixture or by sorbitol supplementation at

either 3 0 or 60 g steer"! day"!; however, there was a

tendency for an interaction between protein source and

sorbitol level for feed efficiency.

Implications

Under stressful conditions when cattle usually lower

their feed intake, a feed additive such as sorbitol,

theoretically should benefit the animal. Likewise, low

ruminally degradable protein has been shown to improve 28 d

31

incoming performance of cattle. However, this study did not

produce data supporting a benefit of sorbitol

supplementation or a low ruminally degradable protein source

to incoming feedlot steers. The lack of response by steers

in this experiment could be attributed to DMI.

32

Table 3.1. Composition of basal diets^.

Item

Readily ruminally degradable

Low ruminally degradable

% %

Steam-flaked grain sorghum Cottonseed hulls Molasses, cane Animal fat, #2 yellow grease Urea Sodium chloride Calcium carbonate Dicalcium phosphate Potassium chloride Trace mineral premix^ Vitamin premix^ AS700 premix^ Soybean meal Escape protein mixture® Sorbitol premix^ Control premixg

Analysis^ DM, % CP, % Ca, % P, % K, % NEm, Mcal/kg NEg, Mcal/kg

39.88 40.08 3.00 1.50 .17 .20 .80 .22 .10 .35 .25 .75 00 .00 .00

1.70

86.52 13.01

.50

.28 1.04 1.69 .86

11

39.88 40.08 3.00 1.50 .17 .20 .80 .22 .10 .35 .25 .75 4.5 5.4 .00

1.70

86 13

1 1

55 02 55 28 00 63 77

^ Dry matter basis. b Contained (ppm) I 1,232, Mn 8,069, Zn 8,409, Cu 827, Co 51, Fe 4,056. c Contained 660,000 lU kg"! vitamin A. ^ Contained 6.16 g aureomycin and sulfamethazine kg"!. 6 Contained 15 % blood meal, 8 % corn gluten meal, 22 % cottonseed meal, 33 % hydrolyzed feather meal, and 22 % meat and bone meal. f Contained 2.0 % mineral oil, 23.6 % ground grain sorghum and 74.4 % sorbitol. g Contained 2.0 % mineral oil and 98.0 % ground grain sorghum. h DM basis except for DM.

33

.

a) o c <c s u 0

<Mh

Q)

4-> 0

i H

feed

> <o

43 tr •H

1

• P

c 0)

*• u 0

(M

to c (0 0) S i r

.nally

rotein

rumi

le p:

^ 4 2 ^ <0

S« 1 ^ «-§

c • H

> . < " . ^ • P Zi 0

0)1 ^ TJ g 1 ^ (0

4J (0 0) v^ EH

• CM

. ro

0) f H A

Hi

^ & 0)

•0

to Q) 3

i H

>

(I4

T3

"5

0 -p • H

4:3 ^ 0 in

43 t/D

X

04

Sorb

4J 0 U a*

(d

w W

0 VO

0 CO

0

0 VO

0

en

0

g 0)

4-> H

•^ ' t

cn

n ^

^ CO

CO

CM

0 H

265.6

60.7

r

"(t •

H VO CM

• CM VO CM

in •

00 in CM

CM

CM

tn 0 r-i X u (d (1) T H «-

Si -P 6 -H 4J ;3 c :? a H

in

•

r CM

•

0 CM

•

0 CM CM

•

8.08

0 'St

.

CO

VO

8.1

0 0

. 00

0 CM

• 00

VO

• r

tr M

^ H

s Q

0 CM

•

^

•

OS VO

•

in CM H

• 1.92

in H

. CM

0

2.1

VO H

• CM

VO H

• CM

in OS

•

H

T3 •0 0)

t r 0) >J <w

• t

* C tJ -H Q (C < 0

00 H

•

0 ON

•

H CM

•

• H H

238

256

1 ^

in CM

H t^ CM

VO CM

H in CM

tJ> ^ ' ^ 0>

^ r^ H

•

4.20

H ON

.

0

3.9

0 t^

. en

0 00

n

00 OS

• m

0) c

rH 00

* CM H

319.4

20.9

n

CM •

0 CM

0

• n CM n

0 •

ON H

00 •

H CO

^ .

•H 4J (d IS 0 • • i H

TJ <d &* 0) c ^ 0) -H CL, (i<

. C <0 0) g

Q) 43 4->

0

V4 0 u u 0)

73 U (d

•0 c (d -p CO

<d

ion.

4J 0 Id

nte

• H

i H 0) > Q)

r H

r-i 0

4J • H 43 U 0 (0

X

Q) 0 M

SOU

c • H

Q) -P 0 VH CI4

43

• to

• H to

i H <d c (d

0 ^

(M

T3 0) > 0 g 0) VH

0)

0) :^

eers

+J to

ine

c < 4 - l

0.-

c 0) cu

4-> C 0) s +J (d (U

4->

U Q) a to u Q) 0)

+J to

0

^ 0)

43 g 3 ^

0

• 73 0) (1)

0

H 1 tr ^

c • H (d tp

0

tJN

^ > 1 0 c 0)

•H 0

• H (M (M Q)

C • H

<d 0

TJ

• 0

•H 4J (d

•H (d 0 • •

TJ 0) (U ELI

(U

3A

8.40

8.30

8.20

^ 8 . 1 0

^ 8.00

7.90

7.80

7.70 1 30

Sorbitol, g/d 60

LD Protein » RD Protein

Figure 3.1. Dry matter intake of steers fed either low ruminally degradable protein (LD Protein) mixture or readily ruminally degradable protein (RD Protein) supplement (SEM = .220).

35

2.5 -r

2.3 --

^2.1 I

< 1-9 +

1.7 --

1.5 30 60

Sorbitol, g/d

LD Protein » RD Protein

Figure 3.2. Average daily gain of steers fed either low ruminally degradable protein (LD Protein) mixture or readily ruminally degradable protein (RD Protein) supplement (SEM = .125).

36

275 J

-O 270 --

^ 265 --o 260 +

^

•1 255 +

•S250 +

^^245 +

O 240 --

: : 235 1 30

Sorbitol, g/d 60

LD Protein * RD Protein

Figure 3.3. Gain efficiency of steers fed either low ruminally degradable protein (LD Protein) mixture or readily ruminally degradable protein (RD Protein) supplement (SEM = 11.1).

37

CHAPTER IV

EFFECTS OF SORBITOL ON SODIUM DEPENDENT

AND SODIUM INDEPENDENT GLYCINE AND

LEUCINE UPTAKE BY CULTURED

BOVINE KIDNEY CELLS

Abstract

A series of in vitro experiments were conducted in the

presence or absence of sodium to determine if sorbitol at

concentrations ranging from 10"^ to 10"!^ molar affects

glycine and leucine uptake by cultured bovine kidney cells.

Sorbitol tended (P = .094) to improve glycine uptake by

cultured bovine kidney cells when sodium was present in the

incubation media. However, addition of sorbitol when sodium

was absent improved (P = .0001) glycine uptake under these

experimental conditions.

Concentrations of sorbitol ranging from 10"^ to 10"!^

molar did not alter sodium dependent (P = .36) leucine

uptake by cultured bovine kidney cells. But as was seen

with glycine, the addition of sorbitol in the absence of

sodium enhanced (P = .0001) leucine uptake. Results

indicate that the presence of sorbitol in the range of 10~!2

molar increases sodium independent glycine and leucine

uptake under conditions of this experiment using cultured

bovine kidney cells.

Introduction

Sorbitol, a natural six-carbon polyalcohol, is found in

various foods such as cherries and apples. This compound is

regarded as a safe for human consumption and is classified

as GRAS (generally recognized as safe) by the Food and Drug

Administration.

Sorbitol has been reported to both improve biological

response of young calves (Daniels et al., 1981; Thivend,

38

1982) and finishing steers (Fontenot and Huchette, 1993) and

bulls (Geay et al., 1992) fed a corn silage-based diet, and

to exhibit numerical improvements in steers fed a steam-

flaked grain sorghum-based diet (Boyles and Richardson,

1993). The response in feed efficiency to sorbitol

supplementation has ranged from +12 and +7 % for experiments

with cattle in the finishing stage fed corn silage-based

diets (Fontenot and Huchette, 1993; Geay et al., 1992,

respectively) and only a +4 % numerical improvement when

finishing steers were fed a steam-flaked grain sorghum-based

diet (Boyles and Richardson, 1993). The reported

improvements in feed conversion along with improved feed

efficiency and rate of gain (+18 and +14 %, respectively) by

bulls when fed a low ruminally degradable protein source

with sorbitol (Geay et al., 1992) lead to the hypothesis

that sorbitol could be altering protein metabolism. The

proteinaceous body tissue is dynamic due to the continuous

state of flux in protein synthesis and degradation (Maynard

et al., 1979). An important component in protein metabolism

involves cellular uptake of amino acids; therefore, it

seemed appropriate to target research in this area. There

are separate amino acid transport systems for large (L-

system) and small (A-system and ASC-system) neutral amino

acids as well as for basic and acidic amino acids (Sato et

al., 1991). Therefore, the objectives of this research were

to determine the effect of sorbitol on glycine and leucine

uptake by cultured bovine kidney cells.

Glycine

Glycine is a non-essential amino acid that is readily

synthesized from common metabolic intermediates in all

organisms (Bender, 1985). Glycine and serine are readily

interconverted, thus glycine can be converted to serine for

gluconeogenesis. Since glycine has the simplest structure

39

(H2NCH2CO2H) of any amino acid and is universal to all

organisms, it was the first amino acid of choice used in

this experiment. Glycine seems to be transported by both a

specific glycine carrier and an imino-glycine carrier that

also carries proline and hydroxyproline, as well as being a

substrate for the ASC-system (Bender, 1985). Maximum

glycine transport is dependent upon the presence of

extracellular sodium ions (Wheeler and Christensen, 1967;

Thomas and Christensen, 1971; Thomas et al., 1971; Hundal et

al., 1989).

Leucine

Leucine is a branched chain amino acid that is wholly

ketogenic and has an important role in mammalian metabolism

as an energy yielding substrate (Bender, 1985). Leucine is

also important as a regulator of protein turnover (Taruvigna

et al., 1979). Low levels of leucine have been shown to

stimulate protein synthesis and higher levels have been

shown to inhibit the catabolism of tissue proteins (Bender,

1985). The L-amino acid transport system possesses a

preference for branched, non-polar side chain amino acids

which includes leucine [(CH3)2CHCH2CH(NH2)CO2H] and this

transport system has been reported to be an sodium

independent process (Boerner and Saier, 1982).

Material and Methods

Bovine kidney cells! were purchased and subcultured in

Eagle's medium^ with 10% horse serum^ upon arrival

! ATCC CCL 22 MDBK (NBL), American Type Culture Collection, 12301 Parklawn Drive, Rockville, MD 20852.

2 Basal Medium Eagle, Sigma Chemical Company, P.O. Box 14508, St. Louis, MO 63178.

3 intergen Company, Two Manhattan Road, Purchase, NY 10577.

40

(Appendix A). Cells were incubated in a water-jacketed

incubator at 37 °C under humidified atmosphere of 95% O2

with a constant injection of 5% CO2. Once cells became

confluent, 10 vials containing cells were stored in liquid

nitrogen for future use (Appendix B). Treatment solutions

were applied after cells became confluent in 24-well Costar^

culture cluster flasks. Treatment solutions consisted of

Krebs-Ringer phosphate buffer (KRP) with and without sodium

plus one of the following: unlabeled amino acids (glycine or

leucine) or ([^HJglycine^ or [^H]leucine^) plus sorbitol at

10"4, 10-6, 10-8^ 10"!0, 10"!2, or 10"!^ molar. The final

amino acid concentration was 200 |iM. Composition of the

KRP-buffer was as follows: KCl 6.0 mM, MgS04-7H20 1.2 mM,

KHCO3 2.0 mM, CaCl2 0.5 mM, NaCl 118 mM, D-Glucose 5.5 mM,

Na2HP04 25 mM. Analysis of sodium independent uptake was

facilitated by replacing sodium chloride and sodium hydrogen

phosphate in the KRP-buffer with choline chloride and

choline hydrogen phosphate in equal molarity. Replacement

of sodium chloride with equimolar concentrations of choline

chloride, lithium chloride or potassium chloride has been

reported to result in a reduction of glycine and alanine

uptake by > 90% (Wheeler and Christensen, 1976). This shows

that glycine or alanine transport is not enhanced by the

presence of choline. However, alkali metal ions other than

sodium have been shown to inhibit neutral amino acid

transport while choline had no effect (Thomas and

Christensen, 1971; Thomas et al., 1971). Therefore, the

4 Costar, 7035 Commerce Circle, Pleasanton, CA 94588 8008.

5 [2-3H]Glycine, Amersham Corporation, 2636 South Clearbrook Drive, Arlington Heights, IL 60005.

6 L-[4, 5--^H]Leucine, Amersham Corporation, 2636 South Clearbrook Drive, Arlington Heights, IL 60005.

41

replacement of sodium chloride with choline chloride

provides a method by which the sodium dependence of amino

acid transport can be investigated without interfering with

transport measurements (Hundal et al., 1989).

Treatment solutions were administered following amino

acid depletion according to the methodology of Vadgama

(1989) (Appendix C). The plated cells were washed twice

with warm choline-KRP and depleted of intracellular amino

acids in 1 mL of choline-KRP for one hour at 37 °C in a gas

atmosphere 95% O2 and 5% CO2 before test solutions were

placed in the wells of the culture flasks. Test solutions

(250 jiL) were incubated at 37 °C (humidified atmosphere of

95% ©2 and 5% CO2) while gently shaking culture plates to

avoid unstirred aqueous layers. The uptake incubation was

terminated at exactly two minutes by decanting uptake medium

and immediate addition of one mL of ice-cold phosphate

buffered saline twice for 15 seconds. To facilitate rapid

washing and treatment application, construction of wash

trays and treatment application trays were done according to

procedures of Vadgama (1989) (Appendices E and F).

The intracellular labeled amino acid was extracted

from the cell layer in each well with 220 |iL of 5%

trichloroacetic acid for one hour and 200 |iL of the extract

was counted in 5.0 mL of scintillation fluid in a liquid

scintillation counter. The protein content was determined

using a modification of the methodology of Lowery et al.

(1957) (Appendix D).

Data from 6 experiments for each amino acid were pooled

and analyzed in a randomized block design using the GLM

procedure of SAS (1990) for both the sodium dependent

(sodium present) groups and the sodium independent (sodium

absent) groups. Days in which the 6 experiments were

conducted served as the blocks. The model included blocks

and sorbitol level. Treatment means were separated by the

42

protected least significance difference procedure (Steel and

Torrie, 1980) and all means are reported as least squares

means. Each treatment well (four treatments group"!

experiment"!) was analyzed for protein content after the

radioactivity data was collected in order to normalize the

radioactivity (disintegrations minute"! [DPM]) to mg of

protein contained within each well. The unlabeled amino

acid treatment served as the negative control and its mean

DPM mg"! protein was subtracted from the other five

treatments. Data is reported as DPM per mg of protein.


Sorbitol tended (P = .094) to improve glycine uptake by

cultured bovine kidney cells when sodium was present in the

incubation media (Table 4.1 and Figure 4.1). There appeared

to be a positive response in glycine uptake by cultured

bovine kidney cells when sorbitol was present at 10"!2 molar

(S12) concentration.

The addition of sorbitol when sodium was absent

improved (P = .0001) glycine (Table 4.1 and Figure 4.1). In

the absence of sodium, sorbitol at molar concentrations of

10~!2 and 10"!^ (SIO) enhanced glycine uptake by cells

compared to cells not exposed to sorbitol (controls) (P =

.0001 and P = .0013 for S12 and SIO, respectively).

Leucine uptake data are shown in Table 4.2 and Figure

4.2. Cellular uptake of leucine when sorbitol and sodium

were present was not different (P = .363) from cells

incubated without sorbitol. However, in the absence of

sodium, leucine uptake was improved by the presence of

sorbitol at S12 (P = .0001) and sorbitol at 10"!^ molar

(S14; P = .002).

The mechanism by which sorbitol is altering uptake of

these two particular amino acids is unclear. Sorbitol is

known to exert an osmotic effect in biological fluids

43

(Merck, 1989) until it is metabolized. Sorbitol is most

likely exerting some osmotic effect. This may partially

explain the response elicited by sorbitol at concentrations

above 10"!0 molar on glycine and leucine uptake as exhibited

in Figures 4.1 and 4.2. Perhaps the presence of sorbitol in

the range of 10"!2 molar alters the sodium gradient or

membrane potential, thereby modifying glycine and leucine

transport.

In conclusion, data indicate that the presence of

sorbitol increases sodium independent cellular uptake of

glycine and leucine by cultured bovine kidney cells under

these experimental conditions. Further research is needed

to elucidate the effect exhibited by sorbitol concentration

at 10"!2 molar on glycine and leucine uptake.

Implications

The possibility exists based on these data that

sorbitol alters glycine and leucine transport by cultured

bovine kidney cells. Bender (1985) states that mammalian

amino acid transport is assumed to be similar in other

tissues as it is in the renal epithelium. Therefore, it may

be possible that sorbitol increases skeletal muscle uptake

of glycine and leucine in vivo.

44

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46

4 6 8 10 12

[Sorbitol], lO-molar

•- glycine NA -B- glycine WO

Figure 4.1. Effects of sorbitol on glycine uptake by cultured bovine kidney cells in the presence of sodium (glycine NA; SEM =123.4) or in the absence of sodium (glycine WO; SEM = 60.1).

47

14000 X

13000 --

2 12000 +

E 11000 --

10000 --

9000 6 8 10

[Sorbitol], lO-molar 12 14

-•- leucine NA -B- leucine WO

Figure 4.2. Effects of sorbitol on leucine uptake by cultured bovine kidney cells in the presence of sodium (leucine NA; SEM = 625.2) or in the absence of sodium (leucine WO; SEM = 584.3).

48

CHAPTER V

CLEARANCE OF INTRAVENOUSLY

ADMINISTERED SORBITOL

IN STEERS

Abstract

Ten crossbred steers (365 ± 17 kg) were used to

determine the effect of intravenous administration of

sorbitol. Steers were fed a steam-flaked grain sorghum,

cottonseed hull based diet for 14 d prior to receiving an

intravenous infusion. Steers were not offered feed 24 h

prior to receiving intravenous injections in each of two

experimental periods. Steers were randomly allotted to

treatments (two steers per treatment) and were intravenously

infused. Treatment solutions were: physiological saline

(.9% sodium chloride), glucose, sorbitol, propionate or

sucrose in 50% solutions (except saline) at 2.2 g kg"!

metabolic weight. The injection site was the jugular vein.

Serial blood samples were collected at the following times

(T) : 0, 2, 30, 60, 120, 240 and 360 minutes.

Sorbitol, intravenously infused, produced a 62%

increase (P = .008) in plasma glucose 120 minutes post

infusion compared to saline infused steers (122.7 vs. 76.3

mg dL"!) and a 22% numerical improvement (P = .19) over

steers infused with propionate (122.7 vs. 100.8 mg dL"!).

Sorbitol infusion produced a slight increase in plasma

sorbitol concentration to a peak of 2.94 mg dL"! 30 min post

infusion. The increase in plasma sorbitol over (P = .01)

saline infused steers was exhibited at 30 and 60 min post

infusion (.23 vs. 2.94; .35 vs. 2.73 mg dL"! foj- saline and

sorbitol infused steers at 30 and 60 min, respectively). In

summary, intravenously administered sorbitol was rapidly

cleared and elicited an increase in plasma glucose in

steers.

49

Introduction

Sorbitol, a polyhydroxy alcohol, has been shown to

improve performance by bovine animals ranging from young

calves to bulls and steers of market weight (Daniels et al.,

1981; Thivend, 1982; Bauchart et al., 1985; Thivend et al.,

1984; Geay et al., 1992; Fontenot and Huchette, 1993).

Recent work reported by Fostier (1992) showed that

sorbitol when administered in the drinking water to Holstein

cows at a slaughtering facility reduced (P < .05) the

percentage of dark cutting carcasses by 30% when compared to

cows not receiving sorbitol. Fostier (1992) also reported

that three kg of sorbitol per animal supplied in the

drinking water after induced stress increased (P < .03) the

glycogen level contained in biopsies from the longissimus

muscle obtained from young Holstein bulls. Shaw (1974)

postulated that sorbitol follows the same metabolic pathways

in ruminants as in non-ruminants. In non-ruminants the

metabolism of sorbitol depends primarily on a hepatic

enzymatic process. Fructose was shown to be the chief

product of the enzymatic oxidation of sorbitol leading to

glucose (Blakley, 1951; Seeberg et al., 1955).

Sorbitol administered intravenously to dogs (Todd et

al., 1939) and rabbits (Seeberg et al., 1955) has resulted

in the development of only moderate levels of sorbitol in

the blood, but caused a prompt elevation of blood reducing

sugar. Since sorbitol dehydrogenase, a hepatic enzyme, is

commercially available prepared from sheep liver and is used

as a diagnostic tool in liver disorders (Asada and Galambos,

1963; Wiesner et al., 1965), one could postulate that

sorbitol metabolism is similar in ruminants. Therefore, the

objective of this research was to determine the clearance

and possible glucogenic property of sorbitol in steers.

50


Ten crossbred steers (3 65 ±17 kg) were fed a steam-

flaked grain sorghum, cottonseed hull based diet (Table 5.1)

for 14 d prior to receiving an intravenous infusion. Steers

were housed in one pen, fed once daily and had ad libitum

access to clean fresh water. Steers were not offered feed

24 h prior to receiving intravenous injections. Steer were

randomly allotted to treatments (two steers per treatment).

Steers were intravenously injected with one of five

treatment solutions at a rate of 2.2 g kg"! of metabolic

weight. Treatment solutions (pH, 7.0) were: physiological

saline (.9 % sodium chloride), glucose!, sorbitol^,

propionate^ (500 mL of propionic acid plus sodium hydroxide

to adjust pH, diluted to 1000 mL) or sucrose^ in 50 %

solutions (except saline). The injection site was the

jugular vein. The time required for the infusion was

approximately five minutes. After a fourteen d adjustment

period, steers were randomly assigned to treatments and the

experiment was repeated.

The procedure for intravenous treatment administration

was as follows: the necks of steers were disinfected with a

chlorhexidine diacetate solution. A 12 gauge needle was

inserted into the jugular vein and the initial blood sample

was obtained. A sterile catheter^ (inside diameter: 1.57

! Dextrose. Fisher, P.O. Box 1307, Houston, TX 77251.

2 Supplied by Rocjuette FRERES, Gurnee, IL.

3 Propionic Acid, Sigma Chemical Company, P.O. Box 14508, St. Louis, MO 63178. pH adjusted with sodiun hydroxide pellets, Fisher, P.O. Box 1307, Houston, TX 77251.

4 Baxter Scientific, P.O. Box 534032, Grand Praire, TX 75053.

5 Clay Adams, Division of Becton Dickinson and Company, parsippany, NJ 07054.

51

mm; outside diameter: 2.08 mm) was inserted through the

needle into the vein. After inserting approximately 38 cm

of the catheter into the vein the needle was removed and the

treatment solution administered. The catheter was then

flushed with heparinized physiological saline and plugged

with a three mL syringe. Sufficient length of the catheter

tubing was glued along the neck (approximately 30 cm) with

livestock identification tag cement^ and taped to the

forehead of each steer.

After the first two blood samples were collected each

steer was removed from the hydraulic squeeze chute and

placed in an individual holding stanchion for 6 h. All

blood samples except the initial sample were collected post

infusion using the catheter. Following each sample

collection, 10 mL of heparinized physiological saline was

flushed through the catheter to prevent blockage. Prior to

each blood collection, the heparinized saline along with

some blood (5 mL) was removed and discarded. Samples were

collected, placed in 15 mL polystyrene centrifuge tubes

containing sodium-fluoride, potassium oxalate mixture (2.5

mg sodium fluoride and 2 mg potassium oxalate per mL of

blood) and immediately placed on ice until plasma

separation, then stored at -80 °C until analyzed.

Plasma was analyzed for glucose^, sorbitol (Bergmeyer

et al., 1974), and fructose (modification of the method of

Anderson et al., 1979, Appendix G).

Data were analyzed using repeated measures analysis of

variance in the General Linear Model procedure of SAS

(1990). There were five treatments with four replications

(steers) per treatment and blood samples were collected

6 Ruscoe, W.J., Ruscoe Company, Akron, OH 44301.

7 Glucose oxidase method, Sigma Diagnostic Kit No. 510, Sigma Chemical Company, P. O. Box 14508, St. Louis, MO 63178.

52

serially seven times from each steer. Treatment means were

separated using the protected least significant difference

procedure (Steel and Torrie, 1980) when there was no

treatment*time interaction. When an interaction existed,

treatment means were separated using the IML procedure so

the proper error mean square could be used for the

denominator of each F statistic. The F statistic for

orthogonal contrasts was calculated using the subplot error

mean square. The standard errors are reported using both

the main and subplot error terms (Steel and Torrie, 1980).


Average initial plasma glucose concentrations for

steers in this experiment was 73.4 ± 1.99 mg dL"! (Table

5.2). After intravenous infusion, steers receiving glucose

showed an increase (P < .0001) in plasma glucose

concentration to 378.6 mg dL"! nd remained higher (P <

.0001) than steers receiving other infusions for the initial

120 min. Plasma glucose levels returned to baseline

concentrations by 360 min post infusion which were similar

(P = .35) to saline infused steers. Response surface

analysis of the glucose infusion over time (Figure 5.1)

produced a quadratic effect (P < .05).

As depicted in Figure 5.2 the plasma glucose of steers

infused with both sorbitol and propionate began to show a

numerical increase over baseline values within 3 0 minutes.

By 120 min post infusion the steers infused with sorbitol

showed an increase in plasma glucose to 122.7 mg dL"! which

was similar (P = .08) to steers infused with glucose and

higher (P = .008) than steers infused with saline (122.7 vs

76.3 mg dL"!, for sorbitol and saline infused steers,

respectively). This constituted a 62 % increase in plasma

glucose 120 min after infusion of sorbitol. Meanwhile

steers that received propionate tended (P = .14) to exhibit

53

an increase in glucose concentration over saline infused

steers (100.8 vs. 76.3 mg dL"!, respectively). Therefore,

by 120 min the sorbitol infused animals demonstrated a 62%

increase over the saline infused steers and a 22% numerical

increase over steers infused with propionate.

The implication of this significant glucogenic effect

elicited by intrajugular infusion of sorbitol when compared

to saline infused steers coupled with the numerical increase

over propionate lends credence to the possibility of

sorbitol serving as a glucose precursor in ruminants.

Metabolites known to serve as significant gluconeogenic

precursors in ruminants are: (1) propionate, (2) glycerol,

(3) amino acids, and (4) lactate and pyruvate (Bergman,

1970; Exton, 1972; Krebs, 1964; Weiss and Loffler, 1970).

Bergman (1973) states that other glucogenic precursors could

be valerate, ribose, citrate, along with a variety of

organic compounds that are present in the body. Results of

this experiment indicate that sorbitol can serve as a

glucogenic precursor in ruminants.

Plasma from steers infused with saline and sorbitol

were analyzed for sorbitol and fructose. Plasma sorbitol

values are listed in Table 5.3 and are shown in Figure 5.3.

After sorbitol infusion, an increase (P = .01) in plasma

sorbitol concentration over saline infused steers was

exhibited at 30 and 60 min post infusion (.23 vs. 2.94; .35

vs. 2.73 mg dL"! for saline and sorbitol infused steers at

30 and 60 min, respectively). As a comparison, Rerat et al.

(1993) using swine recently reported peak sorbitol portal

blood concentration of 4.76 mg dL"! ^d arterial blood

concentration of .56 mg dL"!. These concentrations were

reached 135 min postprandial when feeding sorbitol as the

disaccharide, Lycasin.

The slight increase in plasma sorbitol in steers

receiving sorbitol can be explained by the rapid hepatic

54

metabolism of sorbitol. Bye (1969) found that sorbitol was

rapidly removed from the blood of 16 humans during and

following intravenous infusion of sorbitol with only 3% of

administered sorbitol escaping via the kidneys. The liver

is a highly vascular organ. In sheep, the fraction of

cardiac output received by the liver is 30-40%. The liver

contains an enormous capillary bed which functions in the

special effects that the hepatic parenchyma cells have on

substances entering the organ (Smith and Hamlin, 1977). The

amount of cardiac output flowing through the liver and the

enormous vascular system it possesses sheds light on the

rapid clearance of sorbitol by steers found in this study,

especially based on a (juick, one time administration of

sorbitol. Bye (1969) reported an increase in human plasma

sorbitol to 350 mg dL"! after 4 hr infusion of 30% sorbitol

solution administered at .6 g min"!. Therefore, to produce

a large increase in plasma sorbitol, sorbitol needs to be

administered at a constant rate over time in order to exceed

hepatic capacity for clearing this compound. *

Plasma from steers receiving sorbitol and saline were

analyzed for fructose concentration. Because fructose is a

metabolic intermediate in the metabolism of sorbitol to

glucose coupled with its reported effect on amino acid

transport, it was deemed important to ascertain plasma

fructose levels. Plasma fructose values are listed in Table

5.4 and shown in Figure 5.4. The mean baseline fructose

concentration for steers used in this experiment was 35.1 mg

dL"! ± 2.88. These values are considerably higher than

those reported in cows (5 mg dL"!; Rice 1953), ewes (3.3 ±

.3 mg dL"!; schreiner et al., 1981), baboons (.6 ± .09 mg

dL"!; Crossley and Macdonald, 1970) and humans (4.7 ± 1.9 mg

dL"!; Yoshioka et al., 1984). Kurz and Willett (1992) have

reported calf plasma fructose concentrations of 63 mg dL"!

55

at birth which declined to undetectable levels by 14 h after

birth.

Neither glucose nor sorbitol when added to standard

solutions in this analysis affected fructose determination

(data not presented). However, the possibility of other

serum components affecting fructose determination can not be

ruled out. Based on the difference between total reducing

sugar (90 - 110 mg dL"!) and glucose (65 - 70 mg dL"!)

concentrations found in lactating dairy cows (Willett,

1993), the fructose baseline concentration of 35.1 mg dL"! ±

2.88 could be attributing to the difference in total blood

reducing sugar and glucose. Further investigation is needed

to adequately document steer plasma fructose concentrations

in this experiment.

Plasma fructose levels were similar for saline and

sorbitol infused steers until 120 min post infusion (Table

5.4). At 120 min the plasma fructose of saline infused

steers were higher (P = .002) than steers that received

sorbitol (44.6 and 27.7 mg dL"!, respectively). At 240 min

plasma fructose were similar (P = .31, 24.5 and 29.8 mg

dL"!; for saline and sorbitol infused steers, respectively).

Then by 360 min post infusion, steers that received saline

possessed higher (P = .03) plasma fructose levels than those

steers infused with sorbitol (34.2 and 22.7, respectively).

In conclusion, only glucose and sorbitol when

intravenously administered produced statistical increases (P

< .05) in steer plasma glucose. Sorbitol was rapidly

cleared and elicited an increase in plasma glucose.

Therefore, data indicates that sorbitol possesses a

gluconeogenic property in steers.

Implications

Intravenous administration of sorbitol in beef steers

increases plasma glucose concentrations. These data support

56

earlier work of Bye (1969) in humans, Seeberg et al. (1955)

in rabbits and Todd et al. (1939) in dogs and also exhibits

that sorbitol when infused intrajugularly in steers

increases plasma glucose. Results of this experiment

indicate that sorbitol can serve as a glucogenic precursor

in ruminants.

57

Table 5.1. Composition^ and analysis of basal diet.

Item Percent




Urea .10

Molasses, cane 1.00

Calcium carbonate 1.10


Salt .18



Nutrient Analysis^

DM, % 86

CP, % 10.01

Ca, % .55

P, % .25

K, % *.83

NEm, Mcal/kg 1.56

NEg, Mcal/kg -75

^ Dry matter basis.


51, Fe 4,056. c Contained (lU/kg) Vitamin A acetate 634,480, Vitamin D

63,448, Vitamin E 1,813.

d Calculated on a DM basis except for DM.

58

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•H O Q) to O 0) -U Q)

to to O U U 0) u

4-> to to

<d 3 tr 0)

o

43 tr •H 0)

o •H •H O Si <d

0)

g

^1

o to o

<D 0 tJN to 3 0 ^ u 3

f H

CT

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400 T

30 60 120 Minutes

240 360

SALINE SORBITOL

GLUCOSE O SUCROSE PROPIONATE

Figure 5.1. Plasma glucose levels of steers receiving an intrajugular infusion of either glucose, sucrose, sorbitol or propionate in 50% solutions at 2.2 g kg~^ metabolic weight or equal volume of saline (SEM = 11.52).

62

130 T

NJ 120

g110 +

2 100 o Tr 90

e 80

S- 70

60 0 30 60 120

Minutes 240 360

SALINE SORBITOL

oSUCROSE -^ PROPIONATE

Figure 5.2. Plasma glucose levels of steers receiving an intrajugular infusion of either sucrose, sorbitol or propionate in 50% solutions at 2.2 g kg~^ metabolic weight or equal volume of saline (SEM = 11.52). Same data as Figure 5.1 except glucose infusion data are not shown.

63

20 T

"51,15 E

o CA

E CA C9

10 --

5 -

60 120 Minutes

•m- SALINE -& SORBITOL

Figure 5.3. Plasma sorbitol levels of steers receiving an intrajugular infusion of sorbitol in a 50% solution at 2.2 g kg~^ metabolic weight -or equal volume of saline (SEM = .867).

64

50 T

^

E ^ CA

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^ 9 u

sma

08

£

45 --.,

40 --< •

35 -

30 --• •

25 -

20 30 60 120

Minutes 240 360

SALINE SORBITOL Figure 5.4. Plasma fructose levels of steers receiving

an intrajugular infusion of sorbitol in a 50% solution at 2.2 g kg~^ metabolic weight or equal volume of saline (SEM = 5.67).

65

CHAPTER VI

INTEGRATED SUMMARY

Sorbitol, a polyhydroxy alcohol generally regarded as

safe (GRAS) by the FDA, has been reported to increase feed

efficiency by veal calves, finishing bulls, and steers. The

mode of action which elicits this effect in ruminants has

not been clearly defined. Therefore, it was the intent of

this research to identify the mechanism of action of

sorbitol in ruminants.

Two feedlot performance studies were conducted to

examine if various levels of sorbitol affected the feedlot

performance of steers fed a steam-flaked grain sorghum-based

diet. The initial feedlot study examined the effect of

supplementing sorbitol over a prolonged feeding period (119

d; n = 112; 337.3 ± 17 kg). Sorbitol was fed at various

levels: basal (no sorbitol), 30 g steer"^ d"^; a variable

rate (20 g first 28 d, 30 g second 28 d; then 40 g steer"^

d~^ the remaining time on feed); and 30 g steer'^ d~^ only

after d 69. Supplementation of sorbitol did not

statistically improve (P > .05) steer feedlot performance

over the 119 d feeding period. However, throughout the

feeding period, supplementing steers with 30 g of sorbitol

d"^ showed a 3.4% numerical increase in ADG and a 4.0%

numerical improvement in fed efficiency over steers

receiving no sorbitol. Steers receiving sorbitol only for

the final 50 d had lower dressing percent (P < .002) than

other steers. Furthermore, an overall treatment effect

tended (P = .16) to alter the carcass lean color. Steers

receiving 30 g of sorbitol d"l appeared to exhibit (P = .03)

a more youthful bright cherry red color of the carcass lean.

A subsequent 28 d feedlot receiving trial was conducted to

determine the response by newly received steers fed either

66

0, 30 or 60 g sorbitol steer"^ d~l in combination with

either a low ruminally degradable protein source or a more

readily ruminally available protein supplement. Performance

of incoming feedlot steers (262 ± 21.7 kg; n = 260) during a

28 day experiment was not improved by feeding a low

ruminally degradable protein or by sorbitol supplementation

at either 30 or 60 grams steer"^ day~l. However, there was

a tendency (P = .18) for an interaction between protein

source and level of sorbitol for gain efficiency.

Sorbitol has been documented to be glucogenic in

humans, rabbits, dogs and rats. Therefore, the possible

glucogenic property in steers was investigated. Sorbitol

intravenously infused in steers produced a 62% increase in

plasma glucose 120 minutes post infusion compared to saline

infused steers and a 22% numerical increase over steers

infused with propionate. Sorbitol was cleared and produced

a slight increase in plasma sorbitol concentration to a peak

of 2.94 mg dL~^ 30 min post infusion.

Reports that sorbitol improved feed efficiency and

daily gain of bulls when fed a low ruminally degradable

protein coupled with the magnitude of improvement shown in

steers fed a corn silage-based diet lead to the hypothesis

that sorbitol could be involved in protein metabolism.

Therefore, glycine and leucine uptake by cultured bovine

kidney cells was studied. Alterations in the sodium

independent uptake of these amino acids were exhibited by

the presence of sorbitol. Data indicate that the presence

of sorbitol at a concentration of 10~^2 molar increases

sodium independent cellular uptake of glycine and leucine by

cultured bovine kidney cells under these experimental

conditions.

In summary, sorbitol did not improve 119 d feedlot

performance of steers fed a steam-flaked grain sorghum-based

diet. However, steers receiving sorbitol only for the final

67

50 d had lower dressing percent (P < .002) than other

steers. Furthermore, steers receiving 30 g of sorbitol d"^

for 119 d appeared to exhibit (P = .03) a more youthful

bright cherry red color of the carcass lean when compared to

steers not fed sorbitol. The significant increase in steer

plasma glucose produced by intravenously infused sorbitol

points to a mode of action of sorbitol in energy

utilization. This may partially explain the improvements in

performance by ruminants when fed lower energy dense high

corn silage-based diets when compared to higher energy dense

diets based on steam-flaked grain sorghum. Sodium

independent uptake of glycine and leucine by cultured bovine

kidney cells was enhanced by sorbitol at a molar

concentration of 10~^2. therefore, sorbitol could possibly

increase skeletal muscle uptake of these two amino acids.

In conclusion, the mode of action of sorbitol includes

energy utilization and a possible role in amino acid

metabolism.

68

LITERATURE CITED

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74

APPENDIX A

CELL SUBCULTURE TECHNIQUE

1. Remove growth media from culture flasks with pipette.

2. Wash (rinse) twice with phosphate buffered saline (5 mL)

3. Add 2 mL Trypsin-EDTA solution to slightly cover bottom

of dish.

(a) Gently swirl the dish.

(b) Cells will become suspended by gently tapping on

side of dish. Be careful of the incubation time or

the Trypsin-EDTA will damage the cell membranes.

4. Add growth media (10 mL) when cells are off dish to stop

Trypsin-EDTA's action of dispersing cells.

(a) Draw and dispense solution several times.

5. Remove contents (cells and growth media) and place in 50

mL centrifuge tube.

(a) Draw and dispense several times to suspend cells.

6. Add additional growth media (5 mL) to solution in

centrifuge tube to aid in wash.

7) Centrifuge (180 x g) for ten minutes at 4 °C.

8) Pour off media.

9) Resuspend in new growth media.

(a) Remove aliquot (400 |il) to count cells.

10. For subculturing into flasks, add 15 mL growth media to

each flask to allow for 4 to 5 days growth of cells.

(a) To each flask add 2 mL of cell/growth media

solution.

(b) Gently swirl.

(c) Observe cells under microscope. Cells should

appear suspended and floating in solution.

11. Place culture dishes in a water-jacketed incubator at

37 °C in 95% O2 with a constant injection of 5% CO2.

75

12. For subculturing into 24 well Costar flasks plate out enough cell-growth media solution to give 250,000 cells mL~^ well-1.

76

APPENDIX B

CELL FREEZING TECHNIQUE

1. Remove growth media from culture flasks with pipette.

2. Wash (rinse) twice with phosphate buffered saline (5 mL).

3. Add 2 mL Trypsin-EDTA solution to slightly cover bottom

of dish.

(a) Gently swirl the dish.

(b) Cells will become suspended by gently tapping on

side of dish. Be careful of the incubation time or

the Trypsin-EDTA will damage the cell membranes.

4. Add growth media (10 mL) when cells are off dish to stop

Trypsin-EDTA's action of dispersing cells.

(a) Draw and dispense solution two or three times.

5. Remove contents (cells and growth media) and place in 50

mL centrifuge tube.

(a) Draw and dispense several times to suspend cells.

6. Add additional growth media (5 mL) to solution in

centrifuge tube to aid in wash.

7. Centrifuge (180 x g) for ten minutes at 4 °C.

8. Pour off media.

9. Mix new growth media plus 5% DMSO (dimethyl sulfoxide) to

equal 20 mL of solution: 19 mL growth media + 1 mL DMSO = 2 0

mL freezing media. Example: For 1:5 split, use only about

16 mL of freezing media to suspend cells. Use 3 culture

flasks.

10. Add 1 mL per chilled cryo-vial.

11. Place cryo-vials in freezing tank (cryo freezing tank

contains isopropyl alcohol at minus 70 °C, this lowers the

temperature 1 °C per minute.

(a) Freezing tank must be at room temperature.

(b) Place tank with vials in minus 70 °C freezing

container for more than 4 h.

(c) Store vials in liquid nitrogen.

77

APPENDIX C

EXPERIMENTAL PROCEDURE FOR TREATMENT

OF CELLS IN COSTAR CLUSTER WELLS

The incubation procedure is a modification of methodology

described by Vadgama (1989). Cells will be cultured in 24-

well Costar culture clusters. Once cells become confluent

the treatments will be administered. Krebs-Ringer Phosphate

buffer (KRP-buffer), pH 7.40 will be used as uptake medium.

The composition of this buffer will be as follows: KCl 6.0

mM, MgS04-7H20 1.2 mM, KHCO3 2.0 mM, CaCl2 0.5 mM, NaCl 118

mM, D-Glucose 5.5 mM, Na2HP04 25 mM. Analysis of sodium

independent uptake is facilitated by replacing sodium

chlorine and sodium hydrogen phosphate in the KRP-buffer

with choline chloride and choline hydrogen phosphate in

equal molarity.

Cells have been incubated in 24 well Costar cluster

flasks as described in Appendix A. Cells are washed with 1

mL choline Krebs ringer phosphate buffer (choline-KRP).

Before removing dishes from incubator two wash trays should

be filled with 2 mL of choline-KRP in each tube. Use one

tray to apply incubation solution (choline-KRP).

The above described steps will be conducted according

to the following steps:

(a) Pour off media in dishes.

(b) Wash one time with choline-KRP (1 mL).

(c) Incubate one hour at 37 °C with 1 mL choline-KRP.

This removes any free amino acids from cell by diffusion

(concentration gradient).

(d) Incubate with 250 ^L test solution for two minutes

while gently swirling the dishes.

(e) Wash twice with ice cold phosphate buffered saline

(one mL).

78

(f) Allow incubation wells to dry (15-30 minutes).

(g) Extract cells by applying 220 |iL 5 % (w/v)

trichloroacetic acid (TCA) for one hour.

(h) Pipette 200 jiL into scintillation tubes,

(i) Add 5 mL scintillation cocktail to each tube and

vortex.

(j) Count activity using scintillation counter.

In short:

1. Wash cells with one mL choline-KRP.

2. Incubate one hour at 37 °C with one mL choline-KRP.

3. Incubate with 250 |iL test solution for two minutes.

4. Wash twice with ice cold phosphate buffered saline (one

mL) .

5. Extract 22 0 |iL 5% (w/v) TCA for one hour.

6. Pipette 200 \xL and count.

79

APPENDIX D

PROCEDURE FOR PROTEIN DETERMINATION

The procedure used is a modification of the Lowery

method (1957) as described by Vadgama (1989). The 24-well

costar flasks were stored at 4 °C until protein

determination. The following methodology is based upon the

amount of protein contained in each well when the cells were

seeded at 250,000 cells per well and reached confluency in

two d.

1. To each well add 200 nL of 1 N sodium hydroxide.

The protein is completely dissolved in approximately 3 0

minutes. After 30 minutes, draw out 100 |iL of solution and

replace with 100 iiL of the 1 N sodium hydroxide thereby

making a 1:1 dilution.

2. Add one mL of Lowery Reagent to each well and

gently swirl the tray and allow to stand for 20 minutes.

The Lowery Reagent is composed of disodium cupric

ethylenedinitrotetraacetate (25 g/L), Na2C03 (20.0 g/L) ,

NaOH (4.0 g/L). If using primary culture refer to Vadgama

for additional component.

3. After 20 minutes, add 100 |iL of Folin-Ciocalteau

reagent diluted with an equal volume of distilled water.

Immediately swirl the cluster tray gently.

4. After 25-30 minutes, read absorbance at 500 nm. If

protein content is above 100 ng then read absorbance at 750

nm. This solution continues to darken over time, therefore

it is suggested that the addition of reagent solutions be

made systematically and absorbance readings recorded

accordingly.

5. Bovine serum albumin is used as the standard

(blank, 25, 50, 75 and 100 |ig) . This assay is linear up to

12 5 |ig of protein.

80

APPENDIX E

CONSTRUCTION OF EXPERIMENTAL WASH TRAYS

(Vadgama, 1989)

Cells in monolayer cultures can be washed rapidly in

the 24-well cluster using the device described by Vadgama

(1989). To construct this wash tray, take the top cover of

a 24-well Costar flasks (Costar 3424) and simply drill 24

12.5 mm holes through it. Smooth the rough edges around the

holes with a file or sandpaper. Now insert into each hole a

plastic test tube, 12 mm in diameter and 75 mm in length

(T1226-42, Baxter Scientific). The tubes are inserted so

that about 3.5 mm of the open end is exposed over the

surface of the tray, and it is then glued into position with

polystyrene cement. Several wash trays should be

constructed so sequential washes are possible. The bottom

of the 24-well culture flask is fitted over the wash tray

containing wash solution. The whole assembly is inverted

and the contents of the wash tray are rapidly transferred

into each well of the Costar tray.

81

APPENDIX F

CONSTRUCTION OF EXPERIMENTAL UPTAKE TREATMENT TRAYS

(Vadgama, 1989)

The construction of this uptake tray enables the

addition of small volumes of test substrate to the monolayer

cultures. This arrangement ensures quantitative transfer,

on subsequent inversion of the whole uptake assembly, of the

contents of each capsule simultaneously onto the adherent

cells in each of the 24 wells of the Costar flask.

The construction procedure is similar to that of the

wash tray (Appendix D), except instead of the long

polystyrene tubes, polypropylene embedding capsules (beam

size 00, Polysciences, Inc., No. 0224) are inserted into 9

mm holes drilled into another cover for the Costar 24-well

flask. The capsules can be attached with cyanoacrylate

adhesive (or any other suitable adhesive) so that the open

end of each capsule extends about 3 or 4 mm inside.

82

APPENDIX G

FRUCTOSE DETERMINATION IN BOVINE PLASMA

(Modification of the method described

by Anderson et al., 1979)

This procedure for fructose determination is based

upon the enzymatic reduction of fructose to sorbitol by

commercially available enzyme (sorbitol dehydrogenase, EC

1.1.1.14; Sigma Chemical Co., St. Louis, MO). The

concomitant oxidation of NADH to NAD provides a means of

determining fructose concentration using the conventional

two-point kinetic assay at 340 nm.

The following methodology was derived based upon the

method by Anderson et al. (1979).

1. Mix 300 laL of plasma with 300 |iL of distilled water.

2. Place in a boiling water bath for seven minutes.

3. Centrifuge for 30 minutes (could be modified as long as

denatured proteins are removed).

4. Dilute 250 |iL of supernate with 250 |iL of water and

centrifuge for an additional 15 minutes.

5. Add supernatant to reaction mixture.

6. Reaction mixture contains: 100 |iL of Na2P04*7 H2O (1 M,

pH 6.8), 150 |iL sorbitol dehydrogenase (100 ng of

protein/mL; final concentration of 4.5 Units), 300 |iL of

distilled water, and 300 nL of sample.

7. Zero spectrophotometer at 340 nm to this solution before

adding 150 |iL of NADH (32 mM) .

8. After three minutes from adding NADH to reaction mixture,

read absorbance (340 nm), and again at 23 minutes.

9. Compare total change in absorbance at 340 nm with that of

fructose standards.

10. Report data as change in absorbance per minute.

83

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