TECHNICAL NOTES 991

ages, it was concluded that transferrin type remains constant during the early life of an animal and there is little reason to suspect changes in later life. There is also indication from the gene frequencies of 22 male and 39 female calves that sex of calf does not influence transferrin type.

W. H. RAuscH T. M. LUDWICK

AND D. F. WESELI Department of Dairy Science Ohio State University, Columbus

REFERENCES

(1) AS~TON, G. C. 1958. Genetics o2 Beta-Glob- ulin Polymorphlsm in Cattle. Nature (Lon- don), 182. 370.

(2)AsHTox, G. C. 1957. Serum Protein Differ- ences in Cattle by Starch Gel Electro- phoresis. Nature (London), 180: 917.

(3) tIoJOAARD, M., MOUSTGAARD, J., AND MOL- LEa, I. 1960. Serum Types in Cattle. Fur- ther Investigations. Aarsberetn. Ins*. Steri- ]itetsforskn. K. Vet.-og Landbohoj sk. (kbh.), 99.

(4) ORNSTEIN, L., AND DAVIS, ~. 1962. Disc Electrophoresis. Printed by Distillation Products Industries (Division of Eastman Kodak Company) prior to journal publi- cation.

(5) ]:~AUSCH, W. H., LUDWICK, T. M., AND WESELI, D. F. 1965. The Determination of Bovine Transferrin Types by Disc Electrophoresis. J. Dairy Sci., 48: 720.

(6) SCHI~ID, D., AND BUSCttMANN, H. 1961. Evi- dence of Blood-Group Factors and Serum Transferrins in Fetal Blood of Cattle. Z. Immunitol., 122: 233.

A C T I O N OF CHL01~AL H Y D R A T E ON R U M E N M I C R O O R G A N I S M S

I N V I T R O

Chloral hydrate is one of the therapeutics used in the treatment of the alimentary form of acetonaemia in cattle (13). I t has been found to increase the level of propionic acid in the rumen of cows after oral administration. The mechanism of this action is not known. Per- haps the explanation must be sought in a se- lective influence on the rumeu microorganisms (1). I t is the purpose of this communication to consider this question with regard to the breakdown of two substrates by the rumen microorganisms in vitro.

EXPERI~[ENTAL :PROCEDURE

From rumen fluid, obtained from flstulated cows, inocula were prepared according to the method of Dehority et al. (7) and incubation techniques were those of Burroughs et al. (6) and Bentley et al, (5). When washed cell sus- pensions were used for cellulose breakdown it was necessary to add the growth factors biotin, p-aminobenzoic acid, and valeric acid (5). In experiments with untreated rumen liquor this was not necessary. As a criterion for the ex- tent of breakdown of cellulose and ground hay, the production of volatile fatty acids from these substrates was measured with the methods of Gerritsma (10) and Fcnner and Elliot (8). The mixtures of volatile fatty acids were then subjected to gas chromatography (Research Specialties Model 600; column: chromosorb W, mesh 60./80 charged with 10% monoester of phenylcellosolve and sebacic acid; column tem- perature 110 C) to determine the relative pro- portions of individual components.

RESULTS AND DISCUSSIOI~I

In all incubation experiments, chloral hy- drate or one of the commercial chloral hydrate

derivatives tested, reduced breakdown of puri- fied substances or fodder ingredients. Chloral hydrate concentrations used were based on the practical dose in acetonaemia, viz., 21 g twice a day. For convenient calculation the rumeu volume of cows was estimated to be 100 liter. Final concentrations in vitro of chloral hydrate were 0.42 rag/milliliter.

In most cases inhibition was greatest with chloral hydrate itself. Reduction was not so great with eompounds like chloralose, where the chloral hydrate is linked to a sugar. From these compounds chloral hydrate was slowly liberated, as could be judged with the Fuj i - warn reaction (11, 12). To get a maximal cellulose breakdown, long incubation times up to 17 hr were necessary, because of the long lag times observed with this substratc (4). When hay was used as a substrate, there was a rise in the percentage of propionic acid under the influence of chloral hydrate, resulting ia a small increase in total propionic acid in com- parison with the blanks without chloral hydrate.

ChIoral hydrate itself was partially decom- posed in the medium to chloroform and formic acid; partially it was reduced to trichloroeth- anol, but no trichloroacetic acid was formed. Formic acid can be a naturally occurring sub- stance in the tureen (3) ; therefore, it is thought that chloroform (perhaps together with the un- changed chloral hydrate) and, to a lesser ex- tent, trichloroethanol would exert a slight bac- teriostatic a.ction which would result in the inhibition observed. Chloroform might be more toxic to Gram-negative than to Gram-positive organisms because of their high lipid content (9). This would explain the observation that at the end of the incubation the microflora _~n the flasks with chloral hydrate was changed to a chiefly Gram-positive one, whereas in the

992 JOURNAL OF DAIRY SCIENCE

T A B L E 1

Inh ib i t ion of b reakdown of two dif ferent subs t r a t e s by chloral hydra t e in vi tro

Incuba t ion Subs t r a t e * Compound b t ime V F A produced

(mmole8 per (hr) i00 ml)

H a y 7 1.25 ~ .01 ~ H a y fl-Chloralose 7 1.05 ~ .01 H a y Dichloralose 7 1.10 ± .01 H a y 16 5.34+-- .12 H a y Chloral hyd ra t e 16 3.29 ± .04 Cellulose d 17 5.32 ~ .05 Cellulose Chloral hyd ra t e 17 3.40 ± .03

Subs t r a t e s were added a t the level of 1 g per 100 ml of nlediunl. F ina l concent ra t ions of chloral hydra t e were 0.42 rag /mi l l i l i t e r ; chloral hyd ra t e deriva-

t ives were added in equinmlar concentra t ions . ¢ Four repl icates were run on each t r e a t m e n t ; ~ S t anda rd devia t ion of the mean. d Ground filter paper .

T A B L E 2

Effect of chloral hydra t e on to ta l and ind iv idua l f a t t y acids levels fo rmed f rom g round hay by rumen mic roo rgan i sms in vi tro

Tota l f a t t y Molar p ropor t ions " acids fo rmed

Ace ta te P r o p i o n a t e B u t y r a t e Va le ra te (%)

Blank b 54.6 26.8 18.5 .... fl-Chloralose b 53.8 32.0 14.0 .... Dichloralose b 46.5 35.0 18.3 Blank ¢ 58.0 20.2 16.0 5.0 Chloral hydra t e ~ 42.9 44.2 11.8 0.5

(mmoles/lO0 qnl) 1.25 4--- .01 1.05 + .01 1.10 + .0] 5.34 ~ .12 3.29 + .04

Conten t s of f lasks were pooled f rom the four repl icates to run the analysis . b A f t e r 7 hr of i ncuba t ion with washed suspens ions of cells. ¢" A f t e r 16 hr of incuba t ion wi th un t r ea t ed tureen liquor.

b l a n k s t h e r e w a s no c h a n g e in t h e c h a r a c t e r - i s t ic m i c r o f l o r a w h i c h w a s p r i m a r i l y G r a m - n e g a t i v e . No lac t ic ac id cou ld be d e m o n s t r a t e d in t he m e d i a w i t h ch lo ra l h y d r a t e a t t he end o f the i n c u b a t i o n s b y the m e t h o d o f B a r k e r a n d S u m n e r s o n (2 ) . T h i s does n o t r u l e o u t t he p o s s i b i l i t y o f a t r a n s i e n t r i s e in lac t ic a c i d u n d e r the i n f luence o f ch lo ra l h y d r a t e , e spe - c ia l ly in v iew o f the e s t a b l i s h m e n t o f a G r a m - pos i t i ve f lora. F u r t h e r i n v e s t i g a t i o n s a r e car - r i ed o u t to a n s w e r t h i s q u e s t i o n . M e a n w h i l e , i t w o u l d s eem t h a t by se lec t ive ly i n f l u e u c i n g t he r u m e n m i c r o o r g a n i s m s , ch l o r a l h y d r a t e w o u l d a l t e r t he p a t t e r n o f i n d i v i d u a l vo la t i l e f a t t y ac ids p r o d u c e d f r o m h a y in v i t ro .

ACKNOWLEDGMENT

I t h a n k P ro f . Dr. L. Seekles for his advice and s t i m u l a t i n g in te res t and Miss W. Kle in for tech- nical ass is tance .

R . A. Pm~cs L a b o r a t o r i u m v o o r 3 f e d i s c h -

V e t e r i n a i r e C h e m i e U t r e c h t , T h e N e t h e r l a n d s

RE~ERENCES

(1) BAAIJ, P. K. 1959. Enke le Aspec ten van de Pensd iges t i e bi j Runde ren in Ve rband me t

Acetonaemie. (Some Aspec t s of the Rumen Diges t ion in Catt le in Rela t ion to Aceto- naemia . ) W i t h a s u m m a r y in Engl i sh . Thesis , Ut recht .

(2) BARKER, S. B., AND SU~k'CERSON, W. IX. 1941. The Colorimetric De te rmina t ion of Lac t ic Acid in Biological Mater ia l . J. Biol. Chem., 138 : 535.

(3) BARNETT, M. J . F., AND REID, R. L. 1961. React ions in the Rumen. Edward Arnold (Pub l i she r s ) , Ltd. , London.

(4) BENTLEY, O. G. 1959. A Compar i son o f Artif icial R u m e n Techniques . Oklahoma Conf. Radio iso topes in Agr icu l tu re TID- 7578, U.S. Government P r i n t i n g Office.

(5) BR.~TI,EY, O. G., JOHNSON, R. R., HE~SH- BERGER, T. V., CLINE, J . H., AND MOXON, A . L . 1955. Cel lulolyt ic-Factor Ac t iv i ty of Cer ta in Shor t -chain F a t t y Acids for R u m e n Mic roorgan i sms in Vitro. J . Nut r i t ion , 57: 389.

(6) BURROUGHS, W., FRANK, N. A., GERLAUGH, P., AND BETHKE, R. M. 1950. P r e l im ina ry Observa t ions upon Fac to r s Inf luencing Cel- lulose Diges t ion by R u m e n Microorganisms. J . Nut r i t ion , 40: 9.

(7) DEHORITY, B. A., EL-SHAZLY, K., AND JOItN- SON, R. R. 1960. S tudies wi th the Cellu- ]olytic F rac t ion of R u m e n Bac te r ia Ob- t a ined by Dif fe ren t ia l Cen t r i fuga t ion . J . A n i m a l Sol., 19: 1098.

TECHNICAL NOTES 9 9 3

(8) FENNER, H., AND ELLIOt, J. M. 1963 Quantitative Me~hod for Determining the Steam Volatile Fatty Acids in Rumen Fluid by Gas Chromatography. J. Animal Ski., 22 : 624.

(9) GALE, E. F. 1959. Ciba Lectures in Microbial Biochemistry: Synthesis and Organisation in the Bacterial Cell. John Wiley & Sons, Inc., N.Y.

(10) GERI~ITS~A, K. W. 1954. Quantitatieve Bepaling van de Lagere ve~czuren in Faeces door Verdelingschromatographie. (Quanti- tative Determination of the Lower Fatty Acids in Faeces by Partition chromatog-

raphy.) With a smnmary in English. The- sis, Utrecht.

(11)),~ALHOTRA, 0. P., AND ANAND, V. D. 1957. Colorimetric Estima¢ion of Chloral (Hy- drate). J. Indian Chem. Soc., 34: 501.

(12) MEYEI~, A. E., A]ffD PEa-LEE MorI'TEP~, :P~. 1957. Die BestimmUng yon Chloralhydrat und seinen Umsetzungsprodukten in: KSr- perfliiss]gkeiten und Geweben. Arzneimittel Forsch., 7: 195.

(13) SEEKLES, L. 1948. The Biochendcal Ap- proach to Animal Disease. The Fison Lec- tures. II. Gastro-Intestinal Autointoxica- tion in Cattle and Horses. Vet. J., 104: 238.

C O M P A R I S O N OF M E T H O D S F O R H Y D R O L Y S I S OF B O V I N E U R I N A R Y

E S T R O G E N C O N J U G A T E S 1

The accurate measurement of urinary estro- gens ~ depends on the quantitative hydrolysis of conjugated compounds. As reviewed by Mellin et al. (4) alkaline hydrolysis only par- tially cleaves estrogen conjugates, and both estrone and estradiol are partially destroyed. Solvent hydrolysis apparently cleaves only sul- fate conjugates. Acid hydrolysis completely cleaves conjugated estrogens but destroys a majority of the ]7 a-estradiol present (3, 5, 6). Enzyme hydrolysis appears nondestructive, but incompletely hydrolyzes a mixture of conju- gates and may have reduced effectiveness in the presence of certain Jzlhibitors (1). Mellin (3) found that beef liver fl-glucuronidase yielded slightly greater levels of free estrogens than did bacterial B-glucuronidase when both were incubated with bovine urine.

The purpose of this study was to compare acid hydrolysis, enzyme hydrolysis, and an en- zyme-acid hydrolysis sequence for effectiveness in cleaving conjugates of ~C-laBeled estrogens in bovine urine.

EXPERIME~-TAL PROCEDURE

Thirty-six microcuries of ]7 fl-estradiol-4-~4C (New England Nuclear Corp.) were admin- istered intravenously to a nonpregnant yearling heifer in eight injections (4.5 t~c/injection). An aliquot of the collected urine was pooled for this study. The pooled urine was counted for ~'C in a liquid scintillation counter (Packard Instrument Co.) using internal standard tech- niques to correct to disintegrations per minute (dpm) of ~'C.

Enzyme hydrolysis. Commercially available fl-glucuronidase preparations were compared for effectiveness in hydrolyzing the 1'C-labeled

z Journal Paper no. 2550, Purdue University Agricultural Experiment Station, Project 1306.

Trivial names used in this paper are as follows : estrone (3"hydroxy-A~'~'5(~°)-estratrien-17-one) ; 17 a-estradiol (A~"~'5(~°)-estratriene-3;17 a-diol); 17 fl- estradiol (A~'8'5(~°)-estratricne-3,17 fl-diol); estriol (A~.'~.~(~°).estratriene.3.16,17.triol)"

estrogen conjugates. The manufacturers' rec- ommendations regarding pH and hydrolysis temperature were closely followed. The en- zymes compared were a bovine liver preparation (Ketodase, Warner-Chilcott Co.) and a snail preparation (Glusulase, Endo Laboratories). In addition to fi-glucuronidase, Glusulase con- tains phenolsulfatase activity.

After addition of 200 or 300 units of Keto- dase or Glusulase per milliliter of urine, the samples were hydrolyzed for 48 or 72 hr. Duplicate samples were m n for each enzyme at each concentration for each length of time. At the end of the hydrolysis period, each sam- ple was extracted with ether and an aliquot of the extract taken for measurement of 1~C.

Acid hydrolysis. Acid hydrolysis was con- dueted by adding 15 ml of concentrated HC1 to each of three 100-ml urine samples and reflux- Lug for 18 mLU. Two other samples were hy- drolyzed for 1 hr.

After hydrolysis, the samples were cooled and extracted with diethyl ether. A counting aliquot was taken from the ether extract.

Enzyme-acid hydrolysis. Eight samples were enzyme-hydrolyzed in duplicate and extracted as outlined above. After ether extraction, the residual urine was acid-hydrolyzed for 18 rain and the urine re-extracted. The ether extracts were combined and an aliquot taken for radio- activity counting.

All of the ether extracts were reduced LU vacuo and the free estrogens separated and purified as outlined by MellLU et al. (5). The isolated estrogens were quantitatively trans- ferred to counting vials and the ~C activity measured.

RESULTS AND DISCUSSIOI~

No differences were found due to enzyme concentration (200 or 300 units/milliliter) or enzyme hydrolysis time (48 or 72 hr ) ; there- fore, the data fr~)m all samples for each en- zyme were combined for further analysis.

Results of the experiment are summarized in Table 1.