E“—‘ ‘1'" 7.". ,3? 4 l ” dick-.50) f‘a‘ 'tfia p 0 v, .- . ... A _-‘, v..'}‘ "'5???" c”'” ””5 fi;.'..‘"§.$s-ége-o: he‘d-shes I O . A... . $25"; a"? ‘ ..—’- int- iw‘o'u A This is to certify that the thesis entitled Use of Hydrogen Gas (H2) Excretion to Assess Small Intestinal Malabsorption in Calves. presented by Robert E. Holland has been accepted towards fulfillment of the requirements for ”-5. degreein_L9.._An._CJ1'n. SCi. John B. Kaneene Major professor Date July 14, 1986 0-7 639 }VIESI_J RETURNING MATERIALS: Place in book drop to LJBRAfiJES remove this checkout from ”- your record. FINES will be charged if book is returned after the date stamped below. USE OF HYDROGEN GAS (H2) EXCRETION TO ASSESS SMALL INTESTINAL MALABSORPTION IN CALVES BY Robert E. Holland A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Animal Clinical Sciences 1986 ll“ NONI ‘.Kl ABSTRACT USE OF HYDROGEN GAS (H2) EXCRETION TO ASSESS SMALL INTESTINAL MALABSORPTION IN CALVES By Robert E. Holland Small intestinal carbohydrate and protein malabsorption, asso- ciated with increased end and total-expired breath H2 excretion, was demonstrated in experiment 1. Oral administration of lactulose signi ficantly increased (2 < 0.001) H2 excretion over values measured be- fore it was given. Hydrogen excreted after chloramphenicol administra- tion was significantly increased (2 < 0.001) from values measured after feeding milk alone. Concurrently, chloramphenicol administration signi- ficantly decreased intestinal villous length (2 < 0.001), and D-Xylose absorption (3 < 0.05), compared to values before treatment was given. In experiment 2, small intestinal malabsorption and diarrhea oc- curred after inoculation of Cryptgspggigium 52. End and total-expired breath H2 excretion, and fecal production were significantly in- creased (E < 0.001, E < 0.025, and E - 0.06, respectively). These ob- servations were limited to the diarrheal stage (Stage 2). Results of feeding 2 diets on H2 excretion are reported in ex- periment 3. Values measured for end and total-expired breath H2 ex- cretion were significantly higher (E < 0.05) while feeding diet 2 (hay and concentrate) compared to values measured for diet 1 (whole milk). DEDICATION To my parents, Mr. and Mrs. S. A. Holland and my wife, Margo Steele-Holland ii ACKNOWLEDGEMENTS I wish to express my sincere appreciation to Dr. Thomas H. Herdt, my major advisor, for his encouragement, guidance and sup- port during the course of this study. I am grateful to Dr. John B. Kaneene for serving as my major advisor in Dr. Herdt's absence. His persistent guidance and unwavering support was relentless through the completion of this thesis. Appreciation and special thanks are ex- tended to Dr. John C. Baker, Dr. Frederik J. Derksen and Dr. Howard D. Stowe, members of my graduate committee, for their stimulating, yet constructive criticism and editorial assistance during the preparation of this thesis. I am grateful to Dr. Edward C. Mather, Chairman, Department of Large Animal Sciences for securing the funds necessary for this project and for providing laboratory space and animal care facilities. Special thanks are extended to Dr. Richard B. Talbot for his encouragement, assistance and interest, and to Dr. Kent R. Refsal for his knowledge and expertise in statistics and statistical modeling. A special thanks is extended to Dr. John L. Gill, Department of Animal Science, for assistance in the statistical analysis, Dr. S. D. Grimes, Department of Pathology, for critiquing this thesis and Ms. S. Eisenhauer for clerical assistance. iii TABLE OF CONTENTS Page LIST OF FIGURES. . . . . . . . . . . . . . . . . . . . . . . . . . vi INTRODUCTION. . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 EXPERIMENT 1:. . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 Breath Hydrogen Concentration and Small Intestinal Malabsorption in Calves Summary. . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 Introduction. . . . . . . . . . . . . . . . . . . . . . . . . .4 Materials and Methods. . . . . . . . . . . . . . . . . . . . . 4 Experimental Protocol. . . . . . . . . . . . . . . . . . . . . 7 Statistical Analysis . . . . . . . . . . . . . . . . . . . . . 8 Results. . . . . . . . . . . . . . . . . . . . . . . . . . . . 8 Discussion. . . . . . . . . . . . . . . . . . . . . . . . . . .9 Footnotes. . . . . . . . . . . . . . . . . . . . . . . . . . 19 References. . . . . . . . . . . . . . . . . . . . . . . . . . 20 EXPERIMENT 2:. . . . . . . . . . . . . . . . . . . . . . . . . . . . 22 Changes in Breath H2 Excretion With Cryptgspgrigium— Induced Diarrhea in Calves Summary. . . . . . . . . . . . . . . . . . . . . . . . . . . .22 Introduction. . . . . . . . . . . . . . . . . . . . . . . . . 24 Materials and Methods. . . . . . . . . . . . . . . . . . . . .25 Experimental Protocol. . . . . . . . . . . . . . . . . . . . .27 Statistical Analysis. . . . . . . . . . . . . . . . . . . . . 27 iv Page Results . . . . . . . . . . . . . . . . . . . . . . . . . . . 28 Discussion. . . . . . . . . . . . . . . . . . . . . . . . . . 29 Footnotes. . . . . . . . . . . . . . . . . . . . . . . . . . .36 References. . . . . . . . . . . . . . . . . . . . . . . . . . 37 EXPERIMENT 3:. . . . . . . . . . . . . . . . . . . . . . . . . . . . 39 The Effects Of Diet 0n Breath H2 Excretion In Calves Summary. . . . . . . . . . . . . . . . . . . . . . . . . . . .39 Introduction. . . . . . . . . . . . . . . . . . . . . . . . . 40 Materials and Methods. . . . . . . . . . . . . . . . . . . . .40 Statistical Analysis. . . . . . . . . . . . . . . . . . . . . 41 Results. . . . . . . . . . . . . . . . . . . . . . . . . . . .41 Discussion. . . . . . . . . . . . . . . . . . . . . . . . . . 42 Footnotes. . . . . . . . . . . . . . . . . . . . . . . . . . .46 References. . . . . . . . . . . . . . . . . . . . . . . . . . 47 CONCLUSIONS. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48 LIST OF REFERENCES. . . . . . . . . . . . . . . . . . . . . . . . . .50 LIST OF FIGURES Figure Page 1.1 Duodenal cannula: opening for stopper (A), cannula barrel 10 (B), Open lumenal segment (C). Schematic representation of mask, breathing bag, and valve 11 system or measuring total breath hydrogen concentrations. Mean total breath (A) and end breath H2 (B) excreted in ppm 12 for each time. Hydrogen excreted after lactulose was fed to calves was significantly increased (P < 0.001) when compared with that after whole milk was fed to calves in both total breath and end breath measurements. Parts per million (PPM) equals 1 molecule of H2 in 999,999 molecules of air. Mean total breath (A) and end breath H2 (B) excreted in ppm 13 for each time. Hydrogen excreted after whole milk was fed to calves and after chloramphenicol was administered was signifi- cantly increased (3 < 0.001) when compared with that after whole milk was fed to calves and before chloramphenicol was adminis- tered. rts per million (PPM) equals 1 molecule of H2 in 999,999 molecules of air. Jejunal villous length (n-20, r-100) before and after chloram- 14 phenicol was administered. Mean villous length was significantly decreased after chloramphenicol was administered (2 < 0.001). Plasma D-xylose concentrations in milk-fed only calves (group l)15 and milk-fed, chloramphenicol-treated (group 2) calves. The chloramphenicol-treated calves absorbed significantly less D-xylose at 60, 90, and 120 minutes (2 < 0.05). Schematic drawing of rectal cannula and fecal collecting bag. 30 Mean end-expired breath H excretion. Mean end breath H2 31 excreted during Stage 2 by the experimental calves was signi- ficantly higher (p < 0.025) than other stages. Mean total-expired breath H2 excretion. Mean total breath 32 H excreted during Stage 2 by the experimental calves was higher (p - 0.06) than other stages. vi 2.4 Mean 1 SEM wet fecal weights for the three stages. *Crypto- 3. 3. l 2 sporidium infection increased (p - 0.06) fecal production during Stage 2. Mean + SEM end-expired breath H excretion. *End breath H eereted for weeks 2 and 3 were significantly higher (P < 0.05) for diet 2. +Overall X i SEM end breath H2 ex- creted among calves was significantly higher (2 < 0.05) for diet 2. Mean + SEM total-expired breath H excretion. *Total breath H2 excreted for weeks 2, 3, and 4 were significantly higher (2 < 0.05) for diet 2. +0verall X 1 SEM total breath H excreted among calves was significantly higher (2 < 0.05) for diet 2. Vii 33 43 44 INTRODUCTION The mucosa of the small intestine of neonatal calves is affected by a variety of infectious and noxious agents. Insults to the mucosa, associated with bacterial, viral and protozoal inflammatory diseases, cause villous atrophy and decreased villous epithelial cell function. Similarly, the oral administration of certain antibiotics and the feed- ing of soybean protein have resulted in villous atrophy as well as crypt hyperplasia. Accompanying villous atrophy is a decrease in brush- border enzymes which are necessary for carbohydrate and protein hydro- lysis(1actase and peptidases) and the efficient operation of the sodium-dependent transport process (Na.K.ATPase). The enzymes are synthesized by and located within the microvillous membrane of in- testinal epithelial cells. Disruption of the mucosa, and associated villous atrophy results in carbohydrate and protein maldigestion, mal- absorption and diarrhea. Dietary carbohydrates and proteins presented to the normal small intestine are enzymatically hydrolyzed into smaller, more readily absorbed components. In malabsorptive conditions, they remain either nondigested or nonabsorbed and pass into the large intestine where they are 1) fermented by colonic bacteria, and 2) increase the osmotic pres- sure Within the intestinal lumen. Colonic bacteria ferment the nonab- sorbed carbohydrates and proteins into organic acids and gases. 1 2 Organic acids increase the osmotic effects of the nonabsorbed carbohy- drates, thereby contributing to the diarrhea and acidosis. The gases produced are carbon dioxide (602), hydrogen (H2) and methane (CH4). A portion of the H2 thus produced is transferred via the blood to the lungs and is excreted in the breath. In humans, determination of pulmonary H2 excretion has been used for the evaluation of small intestinal carbohydrate malabsorption. Under normal conditions, H2 production within the small intestine is negli- gible. With complete absorption of dietary carbohydrates and protein, little change in H2 excretion is observed. However, in small intestinal malabsorptive conditions, nonabsorbed carbohydrates and proteins pass into the large intestine where bacterial fermentation occurs, resulting in increased H2 production. The specific objective of these experiments was to measure end- expired breath and total-expired breath H2 excretion as a means of evaluating small intestinal carbohydrate and protein malabsorption in preruminating calves. The primary objectives were 1) to measure end- expired breath and total-expired breath H2 excretion resulting from induced carbohydrate and protein malabsorption, 2) to determine the changes in breath H2 excretion resulting from Cryptggpgrigigg-induced diarrhea, and 3) to determine the effects of diet on H2 excretion. EXPERIMENT 1: Breath Hydrogen Concentration and Small Intestinal Malabsorption in Calves SUMMARY Breath hydrogen concentrations were measured to assess intes- tinal carbohydrate malabsorption in preruminanting calves. Oral ad- ministration of 1.25 g of lactulose (a nonabsorbable carbohydrate)/kg to calves produced breath hydrogen concentrations significantly higher (P < 0.001) than values determined after calves were fed milk and be- fore the treatment was given. This indicates that, in the calf, fer- mentation of nonabsorbed carbohydrates results in increased breath hydrogen values. To induce small intestinal malabsorption, chloram- phenicol was administered orally at 50 mg/kg, 2 times a day, to 5 calves for 3 days. Before therapy was started, each calf was fitted with a duodenal cannula to facilitate collection of intestinal mucosal biopsy samples during treatment. Chloramphenicol therapy significantly increased (2 < 0.001) breath hydrogen concentrations from those mea- sured after calves were fed milk alone. Concurrently, chloramphenicol administration significantly decreased intestinal villous length (3 < 0.001) and D-xylose absorption (p < 0.05), compared with those values before treatment was given. These results demonstrate that decreased intestinal absorptive capacity is associated with an increase in breath hydrogen concentrations and that breath hydrogen may be useful in evaluating malabsorption in calves with naturally occurring enteric disease. INTRODUCTION Carbohydrates presented to the normal small intestine are enzy- matically hydrolyzed into smaller components, which are readily ab- sorbed. In malabsorptive states, carbohydrates remain either non- digested or nonabsorbed in the small intestine and pass into the large intestine where these are fermented by colonic bacteria.1 The co- lonic bacteria ferment nonabsorbed carbohydrate and/or protein into or- ganic acids and gases. The major gases produced are carbon dioxide (C02), hydrogen (H2) and methane (CH4),1’2 Measurable concen- trations of hydrogen and methane are not found in the atmosphere. In addition, these gases are not products of mammalian metabolism, but are 2-4 A derived entirely from bacterial fermentation within the colon. portion of the hydrogen gas produced in the colon is transferred in the blood to the lungs and is excreted in the breath.5'7 Thus, the mea- surement of expired H2 by the breath H2 test provides a clinical index of carbohydrate malabsorption and lactase deficiency.1’6’10 The purpose in the present study was to describe the clinical usefulness of breath H2 as a means of estimating carbohydrate malab- sorption in healthy preruminanting calves and in calves where malab- sorption was induced with chloramphenicol. MATERIALS AND METHODS Animals - Nine male Holstein calves were obtained at birth from cows in the same herd. The perineal areas and udders of the parturient cows had been washed with disinfectant soap, and the calves were de- livered onto a clean sheet of plastic, removed immediately from the barn, and isolated from other cattle. All calves were fed their dam's 5 colostrum for two days. At the end of the colostrum feeding, each calf was fed raw, whole, bovine milk at a quantity equivalent to 10% of its body weight, divided into 2 equal feedings/day, for the dura- tion of the experiment. The calves were randomly alloted to 2 groups, group I comprised 4 calves, and group II comprised 5 calves. A physical examination, CBC and bovine serum chemistry profile were obtained for each calf. None of the calves used in these experiments showed evidence of systemic disease or diarrhea. During the first week of life, calves in group II were surgic- ally fitted with an open T-shaped intestinal cannula (Fig l). A l-week adjustment period was allowed following surgery. The experi- ment was begun when the calves were 3 weeks of age and weighed an aver- age of 60.15 i 1.37 kg (mean 1 SEM). Measurement techniques - Breath H2 samples were obtained by 2 tech- niques for both groups: total-expired breath and end-expired breath. Total breath H2 samples were obtained by placing a face mask con- b a to a 4-L breathing bag over the exter- nected by a Rudolph valve nal nares and mouth. The mask was held snugly in position until the bag was filled. It was compressed once more and then allowed to re- fill. A 60-ml syringe to which a 3-way stopcock was attached was used to obtain an aliquot of air from the bag. To obtain end breath H2 samples, the skin over the trachea was clipped and aseptically scrubbed. Two percent Lidocaine (10 ml) was infiltrated subcutaneously midway over the trachea. A 1- to 2-cm full thickness skin incision was made into the anesthetized area. A 5.0 cm 6 10-gauge needle was inserted between 2 tracheal cartilage rimgs, and into the lumen of the trachea. Twenty-five centimeters of polyethy- lene tubing0 was passed through the lumen of the needle in the tra- chea to just beyond the bifurcation of the trachea. The lO-gauge needle was removed, and a needle adapter was inserted in the tip of the tubing which was sutured to the skin. At the end of the visible expir- ation, 50 ml of gas was aspirated from the polyethylene tubing. This sample should have had the approximate composition of alveolar gas. A D-xylose absorption test was administered to each calf in both groups. After calves were fasted for 24 hours, D-xylose was adminis- tered orally via a nipple bottle at a dosage of 0.5 g/kg in a 5% aqueous solution. Blood was withdrawn from the jugular vein through a previously placed jugular catheter at 0, 30, 60, 90, 120, 150, 180, 240 and 300 minutes after D-xylose was administered. The heparinized blood was immediately centrifuged, and the plasma was frozen until assayed.11 Each calf in group II was fitted with an open T-shaped duodenal cannula fabricated from Silastic medical-grade tubing.d The cannula was surgically inserted into the descending duodenum approximately 30 cm caudal to the pylorus and exteriorized through the right mid- flank. Biopsy samples of intestinal mucosa were obtained using a suc- tion biopsy instrument.e The biopsy tool was passed through the T- shaped cannulae distally along the intestine for 50 cm. This was done to avoid cannulation effects and to obtain mucosa from the proximal je- junum. The biopsy specimens were placed in 10% buffered formalin until villous length determinations were made. The fixed specimens were stained with 2% new methylene blue and examined under a dissecting 7 microscope equipped with an ocular micrometer. Twenty villi were measured on each specimen.12 gnnnnnnngxnnnln nnnlynig - Total breath and end breath hydro- gen samples were analyzed on a gas chromatographf which contained a solid state detector for the specific analysis of H2. The chromato- graph was supplied with an internal pump which provided room air as the carrier gas. However, a reference gas containing a known concentration of H2 is required for calibration.13 EXPERIMENTAL PROTOCOL Groun 1 - Breath H2 excretion was measured after whole milk feeding and lactuloseg were fed to calves. Hydrogen samples were taken before the morning feeding and at l-hour intervals for the next 6 hours. The calves were fasted for 12 hours, and 1.25 g of lactu- lose/kg was administered orally as a single dose in 2 L of water via a nipple bottle. Lactulose, a nonabsorbable carbohydrate, was adminis- tered to simulate malabsorption. Hydrogen samples were taken for lac- tulose feeding in the same manner as described for milk feeding. D- xylose absorption test was performed on the seventh day. Qggun II - Breath H2 samples and jejunal biopsy samples were obtained in response to whole milk feeding before and after the 3-day chloramphenicol regimen. The first breath H2 test was done on the first day of the experiment. On day 2, the initial mucosal biopsy samples were obtained. Subsequently, chloramphenicol was administered for the next 3 days at 50 mg/kg 2 times a day. At the end of chloram- phenicol adminstration (day 5), additional mucosal biopsy samples were obtained. D-xylose absorption was done on the sixth day. The second breath H2 test was performed on the eighth day. This was done to 8 allow sufficient time for the bacteria to become reestablished and to avoid the H2 produced by D-xylose fermentation within the large intes tine. STATISTICAL ANALYSIS Analyses were done to determine the significance of H2 excretion resulting from lactulose feeding compared with that from whole milk feed- ing in group I and with that from whole milk feeding before and after chloramphenicol was adminstered to group II. Therefore, for H2 excre- tion, only within group comparisons were made. Analysis of variance, us- ing calf, treatment, and time as main effects with an interaction effect of treatment and time, was used.14 Fisher's variance-ratio (F test) was used to determine significance between treatments for each group. To assess the effect of chloramphenicol administration on jejunal villous length, analysis of variance, using calf and time as determinant vari- ables, was used. For this analysis, each calf served as its own control. The F test was used to test differences between villous length before and after chloramphenicol was administered. D-xylose absorption was analyzed by analysis of variance, using a repeat measure split-plot model. Bonferroni-n statistic was used for comparing means.15 Correlation between total breath and end breath H2 excretion was determined by Pearson's correlation analysis. RESULTS finnnn l - Total breath and end breath H2 excretion after whole milk and lactulose were fed to calves are shown in Figure 3. Total and end breath H2 concentrations were significantly increased (p < 0.001) after lactulose was fed to calves when compared with those values after whole milk feeding. 9 annn ll,- Chloramphenicol administration increased breath H2 concentrations over those measured before treatment was given (2 < 0.001) (Fig 4) and decreased both intestinal villous length (2 < 0.001) and D-xylose absorption (2 < 0.05) (Fig 5 and 6, respectively). The severity of villous atrophy (Fig 5) can be associated with the administration of chloramphenicol.16 Although histologic changes were observed in 4 of the 5 calves, it is noted that 3 of the 5 developed diarrhea and 2 had softer stools. Average villous length was longer in calf No. 5 after chloramphenicol administration. Time trend changes on plasma xylose concentrations of groups I and II calves are shown in Figure 6. D-xylose absorption peaked at 90 minutes at a maximal concentration of 66 mg/dl for group I calves and at a maximal concentration of 45 mg/dl for group II calves. The chloramphenicol- treated calves absorbed significantly less D-xylose at 60, 90 and 120 minutes (2 < 0.05). Correlation analysis was done on total breath and end breath H2 responses. Total breath and end breath were highly correlated (r2 - .8118, g - 0.001). DISCUSSION The present experiments have shown that carbohydrate malabsorp- tion in preruminanting calves can be detected by the breath H2 test. The oral administration of 1.25 g of lactulose/kg produced breath H2 concentrations significantly higher than the values obtained after whole milk was fed to calves. The breath H2 response obtained with lactulose administration was indicative of colonic bacterial fermenta- tion of the nonabsorbed carbohydrate. Lactulose (4-0-B-D-galactopy- ranaosyl-D-fructose) is a synthetic, nonabsorbable disaccharide.17’18 10 I27 c m <+-9 A B 3.0 cm c w e—s.o——a C m ._. ————_———_, —'_. ____ __—_ Figure 1.1: Duodenal cannula: opening for stopper (A), cannula barrel (B), open lumenal segment (C). ll // ——Mosk Tubing Rudolph Valve —Breothing Bag (4 L) \lr - I I §-— —C|omp Stopcock Port . ,__.' Figure 1.2: Schematic representation of mask, breathing bag, and valve system for measuring total breath hydrogen concentrations. 12 LWHOLE MILK A "WHOLE MILK 60 r- .- HLACTULOSE H LAC TU LOSE so L - 4o - - I“ C) T’ 5 I J l l l J I a 1 O 60 I20 I80 240 330 420 O 60 I20 I80 240 330 420 MINUTES MEAN H2 EXCRETED IN PPM (” C) ' _._..._. wfi-«r Figure 1.3: Mean total breath (A) and end breath H2 (B) excreted in ppm for each time. Hydrogen excreted after lactulose was fed to calves was significantly increased (P < 0.001) when compared with that after whole milk was fed to calves in both total breath and end breath measurements. Parts per million (PPM) equals 1 molecule of H2 in 999,999 mole- cules of air. A ' H BEFORE HAFTER O) C) EXCRETED IN PPM (3 fi m u b 04 (D C) C) r m I 5 I MEAN H2- 13 B ' H BEFORE o—e AFTER Y l l 1 1 0 60 I20 I80 240 350 420 O 60 I20 I80 240 350 420 l l 1 MINUTES 7, __fi_._._—— Figure 1.4: Mean total breath (A) and end breath H2 (B) excreted in ppm for each time. Hydrogen excreted after whole milk was fed to calves and after chloramphenicol was administered was significantly increased (2 < 0.001) when compared with that after whole milk was fed to calves and before chloram- phenicol was administered. Parts per million (PPM) equals 1 molecule of H2 in 999,999 molecules of air. l4 IBEFORE CHLORAMPHENICOL ADMINISTRATION 800 I' CI AFTER CHLORAMPHENICOL ADMINISTRATION 700 - 600 - I": 500 - 400- ;e 2~300- zoo- IOO- / ' CALF No._ 5 6 7 8 9 761ng x :SEM WITHIN CALF AMONG CALVES V.- Figure 1.5: Jejunal villous length (n—20, r-100) before and after chloramphenicol was administered. Mean villous length was significantly decreased after chloramphenicol was administered (3 < 0.001). 15 90 r- 80 L. 70 - so - so - 4-0h mg/dl 30- 20 " , o CHLORAMPHENICOL- TREATED '0 ‘ e MILK-FED l l I l l l I J o 30 so so l20 I50 I80 240 300 Minutes after Treatment _ ,.__ __ —__ m, ___ ._ ......___ _ i - Figure 1.6: Plasma D-xylose concentrations in milk-fed only calves (group 1) and milk-fed, chloramphenicol-treated (group 2) calves. The chloramphenicol-treated calves absorbed sig- nificantly less D-xylose at 60, 90, and 120 minutes (g < 0.05). 16 It had a 2-fold purpose in this experiment. First, it was used to in- duce physiologic malabsorption. It passes through the small intestine nonabsorbed, reaching the colon where it is fermented by colonic bac- teria to short-chain organic acids and gases.6'17 Second, it con- firmed the presence of H2-producing bacteria in the gastrointestinal tract of preruminant calves. In the calves in group II, carbohydrate malabsorption was in- duced by the oral administration of chloramphenicol. The oral adminis- tration of chloramphenicol has been shown to decrease the number and height of the villi and microvilli, resulting in a decrease in the total surface area for absorption within the small intestine.16 In this experiment, chloramphenicol administration induced small intes- tinal malabsorption as observed by the increase in breath H2 excre- tion and decrease in D-xylose absorption. The decreased D-xylose ab- sorption after chloramphenicol was administered was presumably the re- sult of decreased synthesis of mitochondrial proteins which are neces- sary for the efficient operation of the sodium dependent cotransport process.16'19'21 22 and calves23 have Malabsorption and diarrhea in persons been reported in association with the oral administration of antibio- tics. Similarly, factors such as diet changes,24'26 infective 27'28 and agezl"29 have been cited as influencing lactase ac- agents tivity. Lactase activity reflects the degree of villous atrophy. The results of the present study indicate that the malabsorption defect was, in part, attributable to villous atrophy. Variability in the concentrations of H2 excreted by the calves in the 2 groups occurred; it was more pronounced among calves in group 17 II. The first breath H2 test results were higher and less consistent in group II than those in group I. This inconsistency may have been affected by the invasive proce- dures used. The cannulae, necessary for acquiring the biopsy speci- mens, may have affected peristalsis and the bacterial population of the intestine. However, the second breath H2 test results were signifi- cantly increased (P < 0.001) over the first test. This effect was anticipated, since villous atrophy developed as expected with chloram- phenicol administration and was accompanied by malabsorption and an in- crease in H2 excretion. It has been cited that the oral administra- tion of antibiotics will decrease the colonic flora and hydrogen pro- duction.8'30’31 Therefore, a decrease in H2 excretion might have been expected, since chloramphenicol has a broad spectrum of activity against both gram-negative and gram-positive bacteria. In the present experiment, H2 excretion was significantly increased after the anti- biotic was administered. Total breath and end breath H2 excretion were obtained to de- termine which was the most sensitive measure of carbohydrate malabsorp- tion. End breath H2 excretion approximates the H2 concentration in alveolar gas. Total breath H2 excretion reflects dilution by dead space volume within the conducting airways, face mask and gas bag. Solomons32 reported a 30% decrease in H2 concentration with an H2 collecting system similar to the one used in the present experiments. Results from the present study indicate that both total breath and end breath H2 excretions are accurate predictors of carbohydrate malab- sorption in calves. In these experiments, total breath and and breath 18 H2 concentrations were highly correlated, indicating H2 gas dilu- tion due to dead space volume is not a major factor in obtaining reliable data. The present investigation demonstrates that the breath H2 test is useful in evaluating induced malabsorption in the preruminanting calf. The decreased intestinal absorptive capacity is reflective of increased H2 concentrations in the expired air. 19 FOOTNOTES: a Rudolph Valve No. 1400, Han's Rudolph Inc, Kansas City, MO. b North American Drager, Telford, PA. c No. 240 Intramedic Polyethylene tubing (inside diameter, 1.67 mm; outside diameter, 2.42 mm), Clay-Adams, Division of Becton, Dickinson & Company, Parsippany, NJ. d Silastic Medical Grade tubing (inside diameter, 0.95 cm; outside diameter, 1.27 cm), Dow Corning Corp, Medical Products Division, Midland, MI. e 4.7-mm Multipurpose Biopsy tube, Quinton Instruments Co, Seattle, WA. f Model 12 Microlyzer, Quin Tron Instrument Co Inc, Milwaukee, WI. g Lactulose (cephulac syrup), Merrell Dow Pharmaceuticals Inc, Cincinnati, OH. h Anacetin, Bio-Ceutic Laboratories Inc, St. Joseph, MO. 20 REFERENCES 1. Niu H, Schoeller DA, Klein PD. Improved gas chromatographic quantitation of breath hydrogen by normalization to respiratory carbon dioxide. J Lnb glin Med 1979; 94:755-762. 2. Perman JA, Modler S. Glycoproteins as substrates for pro- duction of hydrogen and methane by colonic bacterial flora. Gastgo- gnngnnlngy 1982; 83:388-393. 3. Levitt MD. Production and excretion of hydrogen gas in man. J e 1969; 281:122-127. 4. Ravich WJ, Bayless TM. Carbohydrate absorption and malab- sorption. Clin Gastroenterol 1983; 12:335-356. 5. Kotler DP, Holt PR, Rosewig NS. Modification of the breath hydrogen test: Increased sensitivity for the detection of carbohydrate malabsorption. J Lab Clin Med 1982; 100:798-805. 6. Perman JA, Modler 8, Olson AC. Role of pH in production of hydrogen from carbohydrates by colonic bacterial flora. J Clin Invest 1981; 67:643-650. 7. Solomons NW, Viteri FT, Hamilton LH. Application of a simple gas chromatographic technique for measuring breath hydrogen. J Lab Clin Med 1977; 90:856-862. 8. Stevenson DK, Shahin SM, Ostrander CR, et a1. Breath hydro- gen in preterm infants: Correlation with changes in bacterial coloniza- tion of the gastrointestinal tract. J Pediat; 1983; 101:607-610. 9. Payne DL, Welsh JD, Claypool PL. Breath hydrogen (H ) response to carbohydrate malabsorption after exercise. J Lab Ciin Med 1983; 102:147-150. 10.Lipschitz CH, Irving CS, Gopalakrishma GS, et al. Carbohydrate malabsorption in infants with diarrhea studied with the breath hydrogen test. J Pediatr 1983; 102:371-375. 11. Tietz NW. Enndnnentals 9f Clinical Chemistgy. 2nd ed. Philadelphia: W B Saunders Company, 1976; 1090-1094. 12. Lee FD, Toner PG. 5 a o of mal e. Great Britain: J B Lippincott Company, 1980; 4-17. 13. Solomons NW, Hamilton LH, Christman NT, et al. Evaluation of a rapid breath hydrogen analyzer for clinical studies of carbohydrate absorption. Dig Dis 891 1983; 25:397-404. 14. Gill JL. es and nal sis Px erimen in th ma and Mgdigal_§gienng§. Vol 2, Ames, Iowa: Iowa State University Press, 1978; 169-214. 15. Gill JL. Design and Analysis of Experiments in the Animal and undignl_§gigngg§. Vol 1, Ames, Iowa: Iowa State University Press, 1978. 16. Rollin RE, Levin K, Mero KN, et a1. Structural and functional changes in chloramphenicol-induced malabsorption in calves, in Ennggggingg. 12th World Cong Dis Cattle, Vol I. World Association for Buiatrics. International Congrescentrum R AI, Amsterdam, The Netherlands, 1982; 247-251. 17. Vince A, Killingley M. Wrong DM. Effect of lactulose on ammonia production in a fecal incubation system. Qagtzoenterglogy 1983; 74:629-633. 21 18. Nelson DC, McGraw WRG Jr, Joyuma AM. Hypernatremia and lac- tulose therapy. JAMA 1983; 249-1295-1298. 19. Caspary WF. On the mechanism of D-xylose absorption from the intestine. Qagtzgentegglggy 1972; 63:531-532. 20. Hindmarsh JT. Xylose absorption and its clinical signifi- cance. Clin_§19gngn 1976; 9:141-143. 21. Csaky TZ, Lassen UV. Active intestinal transport of D-xylose. Biocnin Bionnys Ant; 1964; 82:215-217. 22. Jacobson ED, Prior JT, Faldon WW. Malabsorption syndrome induced by neomycin: morphologic alterations in the jejunal mucosa. J Lab glin Mgd 1960; 56:245-250. 23. Huffman EM, Clark CH, Olson JD, et a1 Serum chlorampheni- col concentrations in preruminant calves; a comparison of two formula- tions dosed orally. J Vet Pharmacgl Iner 1981; 4:225-231. 24. Huber JT, Rifkin RJ, Keith JM. Effect of level of lactose upon lactase concentrations in the small intestine of young calves. J_Dni;y_§ni 1964; 47:789-792. 25. Kilshaw PJ, Slade H. Villous atrophy and crypt elongation in the small intestine of preruminant calves fed with heated soybean flour or wheat gluten. Rg§_ygn_§n1 1982; 33:305-3081 26. Jacobs KA, Norman P, Hodgson DRG, Cymbaluk N. Effect of diet on the oral D-xylose absorption test in the horse. An_J_yg;_Rg§ 1982; 43:1856-1858. 27. Celeda LC, Fendrich Z, Serrius K, et al. Xylose absorption in normal and diarrhoeic calves. zgn;;nlhl_!gterinn;mgd_£fil 1983; 30:189-194. 28. Woode GN, Smith C, Dennis MJ. Intestinal damage in rota- virus infected calves assessed by D-xylose malabsorption. Vet Reg 1978; 102:340-341. 29. Roberts MC. Carbohydrate digestion and absorption studies in the horse. Res Vet Sci 1975; 18:64-69. 30. Davidson GP, Goodwin D, Robb TA. Incidence and duration of lactose malabsorption in children hospitalized with acute enteritis: study in a well-nourished urban population. J Eedintz 1984; 105:587-590. 31. Gilat T, Ben Hur H, Gelman-Malachi E, et a1. Alterations of the colonic flora and their effect on the hydrogen breath tests. fin; 1978; 19:602-605. 32. Solomons NW. The hydrogen breath test and gastrointestinal disorders. Qomn; The; 1981; 7:7-15. EXPERIMENT 2: Changes in Breath H2 Excretion with Qnynnngnggiginm-Induced Diarrhea in Calves. SUMMARY Breath hydrogen gas (H2) concentrations were measured in five healthy (control) calves and in seven calves incoulated with ern 2- §nnxig1nm gn.(experimental). Each calf was fitted with a rectal can- nula and a fecal collecting bag. The control calves were maintained free of detectable infectious agents. The experimental calves were given 107 to 108 QIXREQEEQIIQIEE oocysts orally in one dose. Time trend changes in H2 excretion after feeding and changes in the weight of feces produced were measured at three stages: before QEXEEQEEQII' ninm inoculation (Stage 1), three days after the onset of diarrhea (Stage 2), and after resolution of diarrhea (Stage 3). There was a treatment-by-stage interaction, where end-expired breath and total-ex- pired breath H2 concentrations were significantly increased (P < 0.001 and 2 < 0.025, respectively). Mean end breath and total breath H2 excreted by the experimental calves were higher (2 < 0.025 and 2 - 0.06) for Stage 2, than the same response measured for the control calves. End breath and total breath H2 excreted by the experimental calves for Stages 1 and 3 did not differ (2 > 0.1) from the controls either within treatments or between stages. Cryptosnoridium infection increased fecal production (2 - 0.06) during Stage 2. The results indicate that Cryntosnoridinn infection caused diar- rhea during Stage 2. The increased H2 production was the result of 22 23 decreased small intestinal absorption of carbohydrates and proteins with the concurrent colonic fermentation of the malabsorbed products. 24 INTRODUCTION Qnyngngnngiginn gnn., an enteropathogen, has been cited as a cause of diarrhea in several animal species and man.1'13 In young calves it most commonly affects the distal small intestine, primarily the mid-to-lower jejunum and ileum.1'10 Lesions in the large intes- tine have also been observed.1'2""S Experimentally inoculated calves develop a profuse, watery diar- rhea 2 to 4 days after inoculation 5’6 with concurrent shedding of oocysts and abnormal feces for 5 to 12 days.l"5'8 The infection causes mucosal damage, as indicated by the passage of feces containing mucus, flecks of blood and fibrin.6’8 Histologic changes in the mu- cosa include atrophy, fusion, blunting and distortion of villi and crypt hyperplasia.1’2’l"S Since the villous epithelium contains disacchari- dase and peptidase enzymes,ll‘"l7 disruption of the mucosa by gnynnn- EEQILQIED could result in deficiencies of these enzymes. The decreased enzyme activity, as a result of mucosa damage, directly affects diges- tion and absorption of carbohydrates and/or protein.3'4 Nonabsorbed carbohydrates and proteins pass into the large intestine where colonic fermentation occurs. Products of this colonic bacterial fermentation include organic acids and gases.18'2o The major gases produced are carbon dioxide (C02), hydrogen (H2) and methane (CI-12).]‘8'20 These gases are absorbed into the blood stream and excreted by the lungs. Therefore, measuring H2 excretion pro- vides a means of detecting colonic H2 production and thus, carbohy- drate malabsorption.18’19’21'22 The objective of this study was to determine if erntosnogi- ding infection in calves caused carbohydrate and protein malabsorption. 25 The breath H2 test was used to assess H2 excretion resulting from deceased small intestinal absorption of carbohydrates and proteins. MATERIALS AND METHODS Animal; - Twelve newborn male Holstein calves were used in the experiment. Seven were assigned to the experimental group and five to the control group. The calves were isolated from other cattle and maintained in individual metabolism cages. A physical examination, CBC and serum chemistry profile were obtained on each calf. All calves used in this experiment were healthy and did not have any evidence of systemic disease. Each calf was fitted with a rectal cannula and a fecal collecting baga (Fig 1). The feces were collected daily and weighed. Dig; - The calves were fed their dam’s colostrum for two days. Following colostrum feeding, they were fed raw, whole, bovine milk at 10% of their respective body weights. The diet was divided into two daily feedings. W mum; - mm oocysts. which were obtained from a naturally infected calf, were given orally to a newborn calf. Feces containing Cryptospogidium oocysts, as determined by the 23 were collected from this carbolfuschin, direct-staining technique, calf and stored at 4°C in aqueous 2% potassium dichromate in a volu- metric ratio of 2:1 (dichromate to feces) until required for inocula- tion. Immediately prior to inoculating the experimental calves, the fecal-dichromate solution was centrifuged and the fecal pellet was added to 8% peracetic acid at 1 part fecal suspension to 10 parts per- acetic acid. The feces were exposed to this solution for 30 minutes, 26 which included centrifugation time. The suspension was washed three times with saline and resuspended in phosphate buffered saline before administration. Each calf was given 107 to 108 oocysts in milk via a nipple bottle. Oocysts were quantified by direct microscopic techniques. ugnnnggmgnn_nggnn1gng§ - End-expired breath H2 samples were collected at the end of visible expiration by way of polyethylene tub- ingb that was placed into the trachea. Twenty-five centimeters of polyethylene tubing were aseptically placed into the trachea and was passed to a point just beyond the bifurcation of the trachea. A needle adapter was inserted and the polyethylene tubing was sutured to the skin. A face mask connected by a Rudolph valvec to a 4-L breathing bagd was used to collect total-expired breath H2 samples. The mask was held over the mouth and nares until the collapsed bag was filled. The bag was compressed once more, then allowed to refill. Hydrogen samples were drawn from the 4-L bag and polyethylene tubing into 60 ml syringes equipped with three-way stopcocks. Hydrogen samples were col- lected before the morning feeding and at 60, 120, 180, 240, 330 and 420 minutes after feeding. Hydrogen samples were collected for each time period for each stage for the two groups. flyggnggn nnalysig - Hydrogen content of each sample was determined by a gas chromatographic technique.24 Egnnl gnllggninn - Each calf was fitted with a rectal cannula. The cannulae were fabricated out of 20 ml syringe cases and rubber tub- ing. They were surgically placed into the rectum. Each calf was given 0.1 mg/kg xylazinee 1M and caudal epidural anesthesia was achieved 27 with 2 m1 of 2% lidocaine hydrochloride.f Ten simple interrupted su- tures were placed circumferentially around the anus. The sutures ex- tended through the skin and external anal sphincter muscle into the rubber tubing surrounding the cannula. Umbilical tape was used to add additional support dorsally. It was tied to a tension-supported hori- zontal mattress suture placed over the dorsal sacrum. EXPERIMENTAL PROTOCOL Prior to inoculating the experimental calves, the breath H2 test was performed on each calf within both groups. Calves in the ex- perimental group were inoculated with Cryntosnoridium oocysts at 3-4 days of age. Fecal samples were obtained daily from the inoculated calves and examined for the presence of oocysts. Feces from all the calves were periodically collected for bacteriological and virological examination. A second breath H2 test was performed 3 days after the onset of diarrhea in the experimental group and a paired response was obtained from the controls. Following resolution of diarrhea, which was determined by the absence of ngntosnoridium oocysts and the return of normal fecal characteristics, a third breath H2 test was performed on both groups. STATISTICAL ANALYSIS Analysis of variance using a repeat measure split-split plot de- sign25 was used to test effects of treatment, stage and time. Treat- ment effects were tested in the main plot and stage and time effects with appropriate interactions were tested in subplots. The F-test was used to determine the significance of each effect. When a significant 28 effect was identified, Bonferroni n-statistic was used for making com- parisons among means.26 Changes in the weight of feces produced were analyzed by ana- lysis of variance using a repeat measure split-plot design.2S Treat- ments were tested in the main plots. Stage and treatment-by-stage in- teraction were tested in the subplots. The F-test was used to deter- mine the significance of each effect. Extreme variation was observed in the weight of feces produced by the experimental calves during Stage 2. Unequal variances were judged to be an important consideration for the comparison that was to be made between the two groups. Therefore, a t-like test statistic26 for one sided alternates was used to make a single comparison between means for the experimental calves and the control calves for Stage 2, only. RESULTS During the experiment, the control calves did not develop diar- rhea and remained free of detectable infectious agents. After inocula- tion, the experimental calves developed diarrhea and started shedding oocysts 5 to 6 days later. Feces collected from these calves were free of other detectable pathogens for the three stages. Mean end breath Hz (Fig 2) and total breath H2 (Fig 3) con- centrations are shown for the three stages and seven time periods. There was a significant treatment-by-stage interaction for both end breath (P < 0.001) and total breath (B < 0.025) H2 excretion. Mean end breath and total breath H2 excreted by the experimental calves were higher (P < 0.025 and P - 0.06, respectively) for Stage 2, than 29 the same response measured for the control calves. End breath and total breath H2 excreted by the control calves did not change among stages (P > 0.1). End breath and total breath H2 excreted by the ex- perimental calves did not differ (P > 0.1) from control calves at Stages 1 and 3. Also, end breath and total breath H2 excreted by the experimental calves during Stages 1 and 3 did not differ (P > 0.1). Mean i SEM changes in the weight of feces produced by the calves for each treatment during each stage are shown in Figure 4. During Stage 2, mean daily production of feces by the experimental calves ranged from 45.70 to 1839.70 g. Feces produced by the control calves ranged from 44.80 to 127.67 g. szntosnoridium infection increased (2 - 0.06) the weight of feces produced by the experimental calves dur- ing Stage 2 when compared to the control calves. Feces produced by the experimental calves did not change between Stages 1 and 3 (P > 0.1) and feces produced by the control calves did not change among stages (2 > 0.1). DISCUSSION This study quantifies the changes observed in breath H2 excre- tion resulting from Cryptosnoridium- induced diarrhea. The ability of the breath H2 test to detect H2 excretion resulting from small in- testinal malabsorption was demonstrated by the strong treatment-by- stage interaction for both end breath and total breath H2 excretion. This effect was limited to Stage 2. The increased H2 excretion was probably due to the malabsorption and fermentation of lactose, although colonic fermentation of nonabsorbed proteins cannot be excluded as a cause of increased breath H2 excretion. This was expected because the Czyntosnogidium infection caused malabsorption and diarrhea. 30 DRAW STRING SYRI NGE — CASE Ii ‘hgii/ ",_./"“2§7 .j ’ FECAL III 4» COLLECTING £\ BAG Ru BER .‘ TUBING \\ Figure 2.1: Schematic drawing of rectal cannula and fecal collecting bag. 01 O I A O _T N O I W IO MEAN H2 EXCRETED IN PPM w . O I -—-— Figure 2.2: 31 CRYPTOSPORI DI UM CONTROL INF ECTE O D STAGE I I 0 STAGE 2 e A STAGE 3 a l l 1 I I l l 0 60 I20 I80 240 330 420 MINUTES Mean end-expired breath H excretion. Mean end breath H2 excreted during Stage 2 by the experimental calves was significantly higher (p < 0.025) than other stages. 32 60 r CRYPTOSPORIDIUM. NTR INFECTED 50 - CI STAGE I I 0 STAGE 2 9 A STAGE 3 ‘ 4or MEAN H2 EXCRETED IN PPM OJ 0 I l I I I I l I O 60 IZO ISO 240 330 420 MINUTES Figure 2.3: Mean total-expired breath H2 excretion. Mean total breath H excreted during Stage 2 by the experimental calves was higher (p - 0.06) than other stages. 33 900 - CONTROL 800 L CRYPTOSPORIOIUM INFECTED T 700- 600T 500* 400 L BOOF 200 m a a SDMflE SDMHE SDMME I _2 _3 NV: MEAN—"E SEM FECES PER STAGE 1” Figure 2.4: Mean 1 SEM wet fecal weights for the three stages. *Cryntosnogidium infection increased (p - 0.06) fecal production during Stage 2. 34 End breath and total breath H2 excretion did not change among stages for the control calves. Therefore, the breath H2 concentra- tions excreted were probably that of normal colonic bacterial fermenta- tion of the residual products of whole milk digestion. End breath and total breath H2 excreted by the experimental calves for Stages 1 and 3 did not differ from the controls, either within treatments or between stages. The concentration of H2 ex- creted by the experimental calves during Stage 1 was comparable to Stage 3. There was also a concurrent reduction in diarrhea. The re- turn to preinoculation H2 excretion and fecal production indicated a return to normal function. Feces produced by the experimental calves during Stage 2 were dramatically increased over that of the control calves. There were no significant differences observed in fecal production within treatments or between Stages 1 and 3. All seven calves given Qnynnnnnnxininn oocysts developed diarrhea. They remained alert, active and continued to consume their daily allotment of milk. Although all the calves de- veloped diarrhea, variation in the quantity of feces produced occurred. The lack of strong statistical significance for the comparison made be- tween the experimental and control calves is reflective of the unequal variances for the two groups. In this experiment, total-expired breath (R - 0.06) was not as sensitive a measure as end-expired breath (E < 0.025). The concentra- tion of total breath H2 excreted was influenced by the dead space that existed within the Conducting airways and gas collecting system. Hydrogen, measured by the end-expired technique, was obtained at the end of visible expiration. This sample should have contained an 35 approximate composition of alveolar gas, thus avoiding some of the dead space contamination that occurred with the total-expired breath H2 tech- nique. Reduced intestinal absorptive capacity associated with an in- crease in breath H2 excretion in the preruminant calf has been demon- strated.27 In that experiment, malabsorption was simulated by the oral administration of a nonabsorbable carbohydrate and induced by the oral administration of chloramphenicol. The nonabsorbable carbohydrate passed through the small intestine unabsorbed to the large intestine where colonic bacterial fermentation resulted in increased breath H2 excretion. The oral administration of chloramphenicol increased breath H2 excretion over that measured pretreatment. It also reduced intes- tinal villous length and D-xylose absorption. In this experiment, the breath H2 test was useful in evaluating malabsorption and diarrhea resulting from a naturally occurring enteric disease. C t s 0 di infection caused temporary malabsorption and diarrhea as evidenced by the significant increase in H2 excretion and fecal production during Stage 2 of this experiment. 36 FOOTNOTES a Steri-Lok Number 8252 Polyethylene Bags (22.8 cm x 43.1 cm) Medical Products Division/3M St. Paul, MN b No. 240 Intramedic Polyethylene tubing (inside diameter, 1.67 mm; outside diameter 2.42 mm), Clay-Adams, Division of Becton, Dickinson and Co., Parsippany, NJ Rudolph Valve No. 1400, Han's Rudolph Inc, Kansas City, MO North American Drager, Telford, PA e Xylazine, Haver-Lockhart, Bayvet Division Cutter Lab. Inc, Shawnee, KA f Lidocaine Injectable, AMFAC Veterinary Supply Co, Atlanta, CA Q0 37 REFERENCES l. Heine J, Pohlenz J, Moon HW. Enteric lesions and diarrhea in gnotobiotic calves monoinfected with ggyntosporidium species. J Infect Dis 1984; 15027680775. 2. Tzipori S. Cryptosporidiosis in animals and humans. Digge- Diel Rev 1983; 47:84-96. 3. Tzipori S, Angus KW, Campbell I, et a1. Experimental infec- tion of lambs with ngntosnoridinn isolated from a human patient with diarrhea. 9n; 1983; 23:71-74. 4. Tzipori S, Smith M, Halpin C, et a1. Experimental crypto- sporidiosis in calves: Clinical manifestations and pathologic find- ings. Vet Dee 1983; 112:116-120. 5. Moon HW, Bemrick WJ. Fecal transmission of calf crypto- sporidia between calves and pigs. Yet Dagnel 1981; 18:248-255. 6. Pohlenz J, Moon HW, Cheville NF, et a1. Cryptosporidosis as a probable factor in neonatal diarrhea of calves. JAVMA 1978; 172:452- 457. 7. Anderson BC. Location of cryptosporidia: Review of the literature and experimental infections in calves. Am J Veg Bee 1984; 45:1474-1477. 8. Anderson BC. Patterns of shedding of cryptosporidial oocysts in Idaho calves. JAEMA 1981; 178:982-984. 9. Pearson GR, Logan EF. Demonstration of cryptosporidia in the small intestine of a calf by light, transmission electron and scan- ning electron microscopy. Veg Bee 1978; 103:212-213. 10. Meuter DJ, Van Kruiningen HJ, Lein DH. Cryptosporidia in a calf. JAXMA 1974; 165-914-917. 11. Current WL, Reese NC, Ernst JV, et al. Human cryptospori- diosis in immunocompetent and immunodeficient persons. N n e 1983; 308:1252-1257. l2. Wolfson JS, Richter JM, Waldron MA, et al. Cryptosporidio- sis in immunocompetent patients. N Engl J Men 1985; 312:1278-1282. l3. Marcial MA, Madara JL. Dnyngosnozigium: Cellular locali- zation, structural analysis of absorptive cell-parasite membrane-mem- brane interactions in guinea pigs, and suggestion of protozoan trans- port by M cells. Qeeexeeneezelegy 1986; 90:583-594. 14. Argenzio RA. Introduction to gastrointestinal function. In: Swenson MJ, ed. anee' Ebyeielegy ef Doneggie Animale. 10th ed. New York:Cornell University Press, 1984; 262-277, 301-310. 15. Freeman HJ, Sleisenger MH, Kim YS. Human protein digestion and absorption: Normal mechanisms and protein-energy malnutrition. glin_geee;eennegel 1983; 12:357-378. 16. Tobey N, Heizer W, Yeh R. Human intestinal brush border peptidases. Qasgroenterology 1985; 88:913-926. 17. Ruckebusch Y, Dardillat C, Guilloteau P. Development of digestive functions in the newborn ruminant. Ann Rech Vet 1983; 14:360-374. 18. Levitt MD. Production and excretion of hydrogen gas in man. N Engl J Med 1969; 281:122-127. 19. Perman JA, Mod;er S. Glycoproteins as substrates for pro- duction of hydrogen and methane by colonic bacterial flora. Gastre- enterelogy 1982; 83:388-93. 38 20. Bond J H Jr, Levitt MD. Fate of soluble carbohydrate in the colon of rats and man. J glln lnves; 1976; 57:1158-1164. 21. King CE, Toskes PP. The use of breath tests in the study of malabsorption. Qlln Gasggoentenol 1983; 12:591-610. 22. Niu, Hsien-Chi, Schoeller DA, Klein PD. Improved gas chro- matographic quantitation of breath hydrogen by normalization to respira- tory carbon dioxide. J Lab Clin Med 1979; 94:755-763. 23. Heine J. Eine einfache Nachweismethode fur Kryptosporidien in Kot (An easy technique for the demonstration of cryptosporidia in feces). Zentzalbl Veterinarmed |D| 1982; 29:324-327. 24. Solomons NW, Hamilton LH, Christman NT, et al. Evaluation of a rapid breath hydrogen analyzer for clinical studies of carbohy- drate absorption. Dig Dis Sci 1983; 25:397-404. 25. Gill JL. si n and nal sis o x eriment the imal end Medicel Sciences, Vol 2 Ames, Iowazlowa State University Press, 1978; 169-214. 26. Gill JL. Design end Anelysis of Ex eriments n the nimal and Medical Seiencee. Vol 1 Ames, Iowazlowa State University Press, 1978; 64-75, 159-182. 27. Holland RE, Herdt TH, Refsal KR. Breath hydrogen concentra- tion and small intestinal malabsorption in calves. In review. An J Vet Reg 1985. EXPERIMENT 3: The effects of Diet on Breath H2 Excretion in Calves SUMMARY The effects of feeding 2 different diets on end-expired breath and total-expired breath H2 excretion was determined. Five healthy calves were fed only raw, whole bovine milk (diet 1) for 4 weeks. Subsequently, the same calves were fed water, hay and concentrate (diet 2) for 4 weeks. Overall, end breath and total breath H2 excreted while on diet 2 were significantly higher (2 < 0.05) than on diet 1. The results demonstrate that H2 excretion was increased by feeding diet 2. The increased excretion of H2 was probably related to the production and excretion of H2 resulting from bacterial fer- mentation within the rumen and gastrointestinal tract. This effect was complemented by the development of the rumen as a fermentative organ and to the establishment of HZ-producing bacteria within the rumen and gastrointestinal tract. 39 40 INTRODUCTION Breath H2 measurements have been used in the two previous experiments to demonstrate an increase in H2 excretion resulting from diarrhea or induced malabsorption in preruminanting calves. The concentration of H2 excreted was reflective of large intestinal bac- terial fermentation of the nonabsorbed dietary products. Those studies were performed in preruminanting calves fed lactose as the primary carbohydrate. When the preruminanting calf is fed roughage and concentrate, it makes the transition from the monogastric system to that of the ruminant. Therefore, the present experiment was undertaken to deter- mine what effects roughage and concentrate feeding would have on H2 excretion compared to whole milk feeding in normal calves. MATERIALS AND METHODS Animals - Five newborn male Holstein calves were used in the experiment. The calves were obtained from a local dairy at birth. All calves were isolated from other cattle and were maintained in individ- ual stalls. Dlene - The calves were fed colostrum for two days. After colo- strum feeding, raw, whole bovine milk (diet 1) was fed. Whole milk was fed at 10% of the calf's body weight divided into 2 equal feedings per day. Diet 1 was fed for 4 weeks. Diet 2 consisted of water, alfalfa hay8 and concentrate.8 The calves had free choice water and alfal- fa hay. The nonmedicated concentrate was provided at 1.0 kg 2 times a day for 4 weeks. 41 Measurement technlguee - Twenty-five centimeters of polyethylene tubingb were placed into the trachea and passed distally beyond the bifurcation. End-expired gas samples were taken from the polyethylene tubing. This was done to collect end alveolar gas. Total breath H2 samples were collected by using a snug-fitting face mask that covered the external nares and mouth. A nonrebreathing valvec was attached to the face mask and gas collection bag.d The mask was held in position until the bag was filled. The bag was man- ually compressed, then allowed to refill. Sixty milliliter syringes equipped with 3-way stopcocks were used to collect H2 from the poly- ethylene tubing and breathing bag. Hydrogen samples were collected before the morning feeding of milk or concentrate and at 60, 120, 180, 240, 330, and 420 minutes after feeding. Hydrogen content of each sample was determined by a gas chromatographic technique.1 STATISTICAL ANALYSIS Analyses were performed to determine the significance of H2 excretion resulting from whole milk feeding compared to water, alfalfa hay, and concentrate feeding. Analysis of variance using calf, diet, and week as main effects with an interaction effect of diet and week was used. The F test was used to determine significance between treat- ments. When a significant treatment effect was identified, Scheffe's Interval was used to make postdata comparisons.2 RESULTS Each calf was healthy with no evidence of diarrhea for the two 4-week feeding periods. One calf developed a tracheal stricture during 42 week 3 of diet 2 and was removed from the experiment. Therefore, for week 4 of diet 2, data are X i SEM of 4 calves. End breath and total breath H2 excreted for diets 1 and 2 are shown in Figures 1 and 2, respectively. End breath and total breath H2 excreted while on diet 2 were significantly higher (2 < 0.05) than for diet 1. End breath H2 excreted at weeks 2 and 3 were signifi- cantly higher (D < 0.05) than for diet 1. Total breath H2 excreted during weeks 2, 3, and 4 were significantly higher (I < 0.05) than diet 1. Neither end breath or total breath H2 excreted during week 1 for either diet differed significantly (D > 0.05). DISCUSSION The results of this experiment indicate that H2 excretion was increased while feeding diet 2. Calves fed diet 2 excreted signifi- cantly more H2 than the same calves fed diet 1. This effect was ob- served for weeks 2 and 3, and weeks 2, 3, and 4 for end breath and total breath H2, respectively. Hydrogen excreted at week 1 for both diets did not differ. Feeding raw, whole bovine milk (diet 1) resulted in H2 excre- tion at concentrations that did not change among the 4 week period. This is attributable to maintaining the preruminant (monogastric) pat- tern of digestion. In bottle feeding whole milk, the liquid meal by- passes the reticulo-rumen and enters the abomasum.3’4 This is accomp- lished by the reflex-closure of the esophageal groove.3 With the introduction of roughage and concentrate, ruminal de- velopment is accelerated.3’5 The transition from the monogastric system to that of the ruminant depends on the diet provided. The 43 [3 DIET I DIETZ I50 Figure 3.1: 6 _ \\\\§i MEAN :r. SEM H2 EXCRETED IN PPM A O r“ 2 3 4 3?: SEM WEE AMONG K CALVES Mean i SEM end-expired breath H2 excretion. *End breath H2 excreted for weeks 2 and 3 were significantly higher (2 < 0.05) for diet 2. +0vera11 X 1 SEM end breath H2 excreted among calves was significantly higher (3 < 0.05) for diet 2. 01 O EXC R ETED IN PPM 8 Af\\—_l 01-5 00 MEAN '3’. SEM H2 — N O 0 Figure 3.2: 44 DDIET I .DIET 2 \ \\\\\\\\\\ ,, \\\\\\\\\\I-,,—I I 2 3 4 XiSEM WEEKS Among Calves Mean i SEM total-expired breath H2 excretion. *Total breath H excreted for weeks 2, 3, and 4 were signifi- cantly higher (2 < 0.05) for diet 2. +Overall X 1 SEM total breath H excreted among calves was significantly higher (2 < 0.05) for diet 2. 45 rumen, once functional, provides a favorable environment for microbial activity. Dietary carbohydrates such as sugars and starches are de- graded into volatile fatty acids and gases.6'8 The 2 major gases produced are carbon dioxide and methane.7’8 Hydrogen is produced by some organisms and reacts with carbon dioxide to produce methane.7’8 The excretion of H2 at weeks 2,3, and 4 for diet 2 was in- creased over that of diet 1 for the same weeks. The rumen, as an organ of fermentation and storage, serves as a constant supply of feed ma- terials for digestion within the gastrointestinal tract. Microbial fermentation of residual feed materials within the rumen and intestinal tract produced increased amounts of H2. The specific site of H2 production was not determined in this experiment. However, it has been reported that H2 is infrequently found or is present in small amounts in rumen gas samples.8'9 Hydrogen excreted at week 1 of diet 2 did not differ from week 1 of diet 1. Limited rumenal development or the absence of H2 produc- ing organisms might have contributed to this observation since the calves were off milk completely at this time. The two previous experiments have demonstrated that increased H2 excretion occurs when diarrhea or small intestinal malabsorption is present. Those experiments were performed on preruminanting calves fed whole milk. Therefore, lactose was the principle carbohydrate. The results of this experiment demonstrate how high concentrations of H2 can be excreted by clinically normal calves fed hay and concen- trate . 46 FOOTNOTES a 22% (crude protein) concentrate, 15% (crude protein) alfalfa hay ob- tained from MSU dairy facilities. b No. 240 Intramedic tubing, (inside diameter, 1.67 mm; outside diameter, 2.42 mm) Clay-Adams, Division of Becton, Dickinson and Co, Parsippany, NJ. c Rudolph Valve No. 1400, Han's Rudolph Inc, Kansas City, MO. d North American Drager, Telford, PA 47 REFERENCES 1. Solomons NW, Hamilton LH, Christman NT, et al. Evaluation of a rapid breath hydrogen analyzer for clinical studies of carbohy- drate absorption. Dig Dis Del 1983; 25:397-404. 2. Gill JL. Deelgn end Anelysig of Experimente in the Animel_ enn_Megleel_§eleneee. Vol 2, Ames, Iowa:Iowa State University Press, 1978;177-178. 3. Roy JHB. Ine CalfI 4th ed. Boston, MA: Butterworth Publishers Inc, 1980; 201-219. 4. Abe M, Iriki T, Kondoll K, et al. Effects of nipple or bucket feeding of milk substitute on rumen by-pass and on rate of passage in calves. Dr J Nut; 1979; 41:175-181. 5. Ruckebusch Y, Dardillat C, Guilloteau P. Development of digestive functions in the newborn ruminant. Ann Rech Ve; 1983; 14:360-374. 6. Argenzio RA. Digestion and absorption of carbohydrate, fat and protein. In: Swenson MJ, ed. k ' o ome Anlne_31,§__L 10th ed. New York:Cornell University Press, 1984; 301-310. 7. Hobson PN. Microbiology of digestion in ruminants and its nutritional significance. In: Cuthbertson D, ed. Nutrlgion ef W WW en e ef_£ezm_lee§eeeke lst ed. Vol 1. Great BritainzAnchor Press LTD, 1969; 59-85. 8. McDonald IW. Physiology of digestion, absorption, and metabolism in the ruminant. In: Cutherbertson D, ed. Nugzlglon ef MW e S ence Nutr on of Iggnnlngeegeek‘ lst ed. Vol 1. Great BritainzAnchor Press LTD, 1969, 87-148. 9. Pilgrim AF. The production of methane and hydrogen by the sheep. Ane;;el_J_§e1_gee 1948; 1:130-138. 48 CONCLUSIONS End-expired and total-expired breath H2 excretion were mea- sured in these experiments to determine whether H2 excretion could be utilized to monitor malabsorption of carbohydrates and proteins in calves. Lactulose was administered to determine what effects a non- absorbable carbohydrate had on H2 excretion. Hydrogen excretion after lactulose administration was significantly higher than values determined after milk was fed. This indicated that, in the calf, fer- mentation of nonabsorbed carbohydrates resulted in increased H2 ex- cretion. Hydrogen excretion resulting from impaired absorption and de- creased mucosal cell function was demonstrated by the oral administra- tion of chloramphenicol in experiment 1 and the oral inoculation of gnynneenenlglnn oocysts in experiment 2. Chloramphenicol induced vil- lous atrophy, thereby causing small intestinal carbohydrate and pro- tein malabsorption. Villous atrophy was demonstrated by decreased D- xylose absorption and a significant decrease in villous length after chloramphenicol administration. It was shown that calves inoculated with Cryptosporidium en exhibited malabsorption and diarrhea. After erntosnegidium inoculation, there was a significant increase in H2 excretion and fecal production. This effect was transitory, since H2 excretion and fecal production returned to preinoculation values. 49 Feeding hay and concentrate caused significantly more H2 ex- cretion compared to feeding whole milk. The increased H2 excretion was probably related to development of the rumen as an organ of fer- mentation and to the persistent fermentation of residual dietary pro- ducts within the gastrointestinal tract. The assessment of H2 excretion in these experiments demon- strated that increased H2 concentrations were excreted 1) when a nonabsorbable carbohydrate was administered, 2) when small intestinal malabsorption was induced by the oral administration of a noxious drug and the inoculation of a known enteric pathogen, and 3) after feeding a hay and concentrate diet. Therefore, the breath H2 test may be use- ful in evaluating small intestinal malabsorption of carbohydrates and proteins in young calves fed milk alone. However, it is an inappro- priate test to use on calves fed hay and concentrate. The end-expired and total-expired interval sampling techniques were an appropriate means of obtaining H2 samples. Although the face mask used permitted considerable dead space contamination, it provided a crude, yet re- liable means of obtaining H2 samples. The end-expired technique was more cumbersome to use, yet provided more consistent values. LIST OF REFERENCES 1. 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