1142

JOURNAL OF DAIRY SCIENCE (2) BEI',ISADOUi'~ A., PALADIN~S~ O. L.~ AND REID~

contaminant if the acids were separated by the W i s e m a n - I r v i n method. Our experience confirms the statement of Wiseman and I r v i n that one micromole of acid is readily detected visually on this column. Its greater visual sensitivity makes it preferable to the silicic acid column of K e e n e y (5). H o w ever, a recent report by Ramsey (8) describes a silicic acid column which may be used to give good separation of the organic acids in ruminant blood. S. L. SPAHR J. B. M O L T E R
AND 2

J. T. Effect of Level of Intake and Physical Form of the Diet on Plasma Glucose Concentration and Volatile Fatty Acid Absorption in Ruminants. J. Dairy Sci., 45: 1203. 1962'.
(3) EgWlN, E. S., MAI~CO.,G. J-., ANI) EMERY, E. M.

E. M. KESLER D e p a r t m e n t of Dairy Science The Pennsylvania State University University P a r k

REFEt~EI~CES

Volatile Fatty Acid Analyses of Blood and Rmnen Fluid by Gas Chromatography. J. Dairy Sci., 44: 1768. 1961. (4) JoYN~, A. E., JR., K~sL~a, E. M., A ~ tIo~T~g, J. B. Absorption from the Omasum and Subsequent Metabolism of Butyrate and Acetate. J. Dairy SEA., 46: 1108. 1963. (5) I~E~N~;Y, M. Direct Chromatographic Determination of Ca to C,, Fatty Acids in Rumen Fluid. Maryland Agr. Expt. Sta., Misc. Publ. No. 238. 1955. (6) McCAg~'HY, 1~. D. Tracer Studies of the Perfused Rumina and Livers of Goats. Ph.D. thesis, University of Maryland. 1958.
(7) MILLER, B. F., AND VAN SLYKEI, D. D. A

(1) ANNISON, E. F. Studies on the Volatile Fatty Acids of Sheep Blood with Special Reference to Formic Acid. Biochem. J., 58: 670. 1954. 2 Present address : Department of Dairy Science, University of New Hampshire, Durham.

Direct Microtitration Method for Blood Sugar. J. Biol. Chem., 114: 583. 19'36. (8) RA~isz, H. A. Separation of Organic Acids in Blood by Partition Chromatography. J. Dairy Sci., 46: 480. 1963. (9) WISEMAN, H. G., AND IavIN, H. M. Determination of Organic Acids in Silage. Agr. Food Chem., 5: 213. 1957.

TEACHING

AIDS

IN RUMEN

PHYSIOLOGY

While interest is perhaps keener in the chemical constitution of living matter than in its physical makeup, a knowledge of the structural features of what is being analyzed chemically is important. I n animals and plants the chemical constituents are combined and arranged into characteristically larger units for f u r t h e r study. Visual aids for instructional purposes have, for the most part, focused attention on the imp o r t a n t role of providing microscopes, fresh specimens, and illustrative classroom materials for students (1, 2). P e r m a n e n t visual models of biological materials in the experiences of the authors have been virtually nonexistent in certain courses. The following inexpensive procedures were developed for assistance in instructional work. The tureen fistula has played a significant role ill contributing to knowledge of the chemical processes which occur in the runmn. H o w ever, the physical processes, which aid in digestion, have been less observed, because of the difficulty encountered in recording these processes, either by instrumentation or visual observation. The excellent nmvie, The Rumen Story, ~ has been very useful in illustrating various physical aspects o the tureen to students; however, it was felt that prolonged observa~Movie produced by the Ralston-Purina Company, St. Louis, Missouri. 1955.

tions, made inside the rumen of the act of swallowing, saliva secretion, regurgitation, and rumen motility might be more revealing and informative. A n inexpensive, easily manipulated optical instrument has been developed ( F i g u r e s 1 and 2) which enables one to observe visually ninny physical processes occurring in the rumen. Rumen periscope. This instrument is constructed f r o m a 12-in. piece of lucite (plexiglass) whose diameter (od 31/2 in.) is such to pernfit it to pass through the fistula plug (lucite type) to a depth of a p p r o x i m a t e l y 10 in. An oval-shaped m i r r o r is supported in the tube at a 45-degree angle by cementing three small supports to the inside wall. To facilitate cleaning of the mirror, the end of the tube is fitted with a screw-type plug. The light fixture and reflector are fastened to this plug at such an angle as to give nmximum light on the object in direct view of the mirror. The light f r o m a 6- or 12-v battery (lantern type) gives ample illumination when in direct focus with the mirror. Light reflections f r o m the polished lucite are eliminated by painting the tube, except for the area in view of the mirror. F o r best observations, the tureen should be practically empty. I t is necessary to heat the assembled instrument in an oven at 40 C for a period of 10-15 rain before use, to eliminate condensation when placed in the rumen. This practice is repeated whenever visibility is lira-

TECHNICAL NOTES
PLEXIGLASS(LEUCITE) T U B E / /

1143

OVAL SHAPED

M,RROR,/'~/

-'Nq' __~
................................. ~ 12"

MOUNTED AT

A P P R O X I M A T E L Y , " / ~ /

4'5

I / " Y ~ I R R O R I ~ O R D . ~ / _ _ SUPPORT~ T H R E A D E NU~T D

I~111 " (SOLID PLEXIGLASS)

//~"

I
I HOLE FOR LIGHT / ATTACHMENT 8,

Fro. 1.
TUBE EXTERIOR PAINTED BLACK

Fro. 2.
ited because of fogging (every 2 hr). All parts of the rumen can be clearly observed by rotation of the tube inside the fistula plug. Preservation of fetal and new-born ruminant stomachs, tIix (4) developed a method for the preparation of the fetal compound stomach. Using fornmlin, filling it with paraffin and applying several coats of cellulose acetate, the preparation preserves the stomach tissue in its natural state for an indefinite period, lends rigidity to the stomach, and renders it resistant to frequent handling. The bovine fetal stomach of 175-195 days of prenatal development is preferred because of its desirable size. The tissues and vascular system at this stage of development are completely differentiated and well developed, and the proportionate size of the four compartments is approximately comparable to that of the adult ruminant stomach (4). An informative visual aid, with the added features o~ viewing the internal anatomy with resiliency rather than rigidity, is outlined and illustrated (Figure 3). Products of this procedure have been in use for approximately 10 yr with no apparent detrimental results. The procedure has been used with stomachs from sheep, goats, and cattle. Oil of wintergreen in combination with other nmterials was used earlier by Spalteholz (5, 7) in the preservation of embryos. 1. Make an incision into the abdominal cavity. Remove the stomach, leaving as much of the esophagus and duodenum region as possible. 2. Drain the stomach fluids, using manual pressure. I t is advisable to use animals that have not nursed, since it is extremely difficult to manipulate coagulated milk through the pyloris without rupturing the abomasum. 3. Fasten glass tubing into the esophagus and duodenum ends so that there is a passageway for materials to drain. 4. Using a syringe, force a 10% formalin solution through the stomach. Clamp the duodenum end and completely fill the stomach. Clamp off the esophagus. Submerge the filled stomach in a 10% formalin solution and leave the specimen for 24-48 hr. 5. Drain the formalin solution. 6. Using a syringe, force methyl salicylate (oil of wintergreen) through the stomach. Clamp the duodenum ~gain and force methyl salicylate into the stonlach so that all compartments are filled thoroughly. Clamp the esophagus. Complete submerge the filled stomach in the methyl sa]icylate solution. 7. Leave the stomach in the solution until it becomes transparent (4 days-1 wk). Turn the stomach several times each day. S. After the stomach is transparent, drain out all the fluid and attach a constant air pressure to dry the specimen. With the four conlpartments inflated and a small amount of air going through the stonmeh, coat the outside with methyl salicylate at 6 to 12 hr intervals. 9. As soon as the specimen appears hard (2448 hr), remove the air pressure.

1144

J O U R N A L OY D A I R Y S C I E N C E

Fro. 3. The prepared fetal goat stomach of 124 days of prenatal development. Visible are the four anatomically distinct compartments of the ruminant stomach. The esophogeal groove, various pillars of the rumen, honeycomb structures of the reticulum, the lamella of the onlasum, and the many folds of the abomasum can be studied. 10. W i t h a scalpel or razor blade cut sections into the four compartments. Coat the outside with cellulose acetate. I f space permits, label the f o u r compartments. Plastic watch glt~ss method of preserving sections of the ruminant stomach. Some of the older-type specimens laeked color contrast, were distorted by the refraction of their glass containers, were bulky and lacked mobility, and required large storage spaces, l~*Iany of these older specimens differed so nmch from the corresponding fresh speeimens that they were of questionable value for instmetional purposes. Specimens p r e p a r e d by the following method a p p e a r to maintain their color and shape over long periods, are easy to handle and transport, and are easily viewed upon close inspection without marked distortion by the containers. The specimens in F i g u r e 4 are examples of this proeedure and were photographed 7 y r a f t e r their preparation. A. Steps used to fix specimens with Klotz No. 1 fixative (3) 1. The tissue is trimmed so that it is not more than 1 in. in thiekness. 2. The tissue is then plaeed in a container with Klotz No. 1 fixative in an amount approxinmtely five times the volume of tissue as soon as the sections are cut from the stomach. The epithelium of the rumen starts to slough away soon a f t e r death. I t is i m p o r t a n t to take the solutions to the slaughterhouse where the specimens are obtained. 3. The fixative solution used is a modified Klotz Solution No. 1 and is p r e p a r e d as follows : Sodium chloride U.S.P ............. 62.5 Sodium bicarbonate U . S . P ....... 112.5 Sodium sulphate U.S.P. (Anhydrous) .......................... 55.0 Chloral hydrate U.S.P ............ 125.0 Formaldehyde U.S.P. 40% ...... 125 W a t e r ........................................ 11,250 g g g g ml ml

4. The specimen is agitated in the fixarive to wash the excess exudate f r o m the tissue surfaces. 5. The specimen then is allowed to stand at room temperature f o r 1 hr or more in the warm Klotz No. 1 solution. 6. The fixative solution is then replaced by new cold Klotz No. 1 solution and the specimen is placed in the r e f r i g e r a t o r and kept just above freezing during most of the fixation procedure. 7. I f the Klotz No. 1 solution has become cloudy or colored by the following day, it is replaced by fresh cold K l o t z No. 1 solution. This procedure is repeated on suecessive days until the solution remains clear. Ordinarily, specimens require a t least three or f o u r changes of fixative solution while refrigerated. 8. The specimen is then allowed to stand at room temperature f o r 24 hr to see if it maintains its natural color. I f the fixative solution becomes colored at room temperature, the specimen should be placed into fresh fixative solution and re-

TECHNICAL NOTES

1145

FIG. 4. Plastic watch glass method of preserving tissue. A. Rumcn, ]3. Retieulum, C. Omasum, D. Abomasum. turned to the refrigerator. 9. Depending upon the size, type, and condition of the specimen, over-all fixation takes about ten days for the sections. B. Steps used to mount specimens by the negative pressure watch glass method 1. A plastic watch glass and a square of plastic plate as the back plate are used as the specimen container. 2. Cut the plastic with a table saw with a fine-tooth blade. 3. I n the permanent mount a special niGunting fluid (Klotz No. 2) is used and prepared as follows for four specimens: parts Propylene glycol (or glycerine) 6 Sodium acetate ........................ 3 Water ........................................ 10 ml 1,800 900 3,000 The mounting fluid should be thoroughly mixed prior to mounting. The Xlotz No. 1 solution used above for fixing is not a good mounting solution, as the salt present will crystallize at contact surfaces and cause leakage. Also, the Klotz No. 1 solution does not maintain color after fixation is accomplished. 4. Wash and dry the plastic watch glass and back plate thoroughly. Place the plastic watch glass into a pan with enough chloroform to cover the bottom of the container. When the plastic edge becomes soluble, hold this side up and place the speeinmn into the watch glass. Be careful that the specimen does not come in contact with the edge. Then place the plastic cover plate over the watch glass and apply pressure. The soluble plastic and negative pressure will fuse together, making an

1146

JOURNAL OF DAIRY SCIENCE

a i r - t i g h t seal. This will be shown b y a clear wide space (the thickness of the w a t c h glass) a r o u n d the w a t c h glass where the f u s i o n took place. 5. D r i l l a hole t h r o u g h the p l a s t i c back. 6. U s i n g a syringe, i n j e c t the fixing solution ( K l o t z No. 2) into the w a t c h glass. 7. W h e n the glass is full, p u t a small cork in the hole. 8. A few days later, remove the cork a n d place a few d r o p s of c h l o r o f o r m a r o u n d the hole. Then p u t a small piece of plastic over the hole. W i t h an eye d r o p p e r a d d c h l o r o f o r m a r o u n d the piece of plastic a n d a p p l y m a n u a l pressure. R e p e a t this p r o c e d u r e several times. 9. The specimen m o u n t s are now p e r m a nent. Label the mounts.

J . L. ALBRIGHT ~

C. L. DAVIS D e p a r t m e n t of D a i r y Science U n i v e r s i t y of Illinois Urbana

AND

T. ]:I. BLOSSER D e p a r t m e n t of A n i m a l Sciences Washington State University Pullman

ACKNO~rLEDGMENT

Preset'ring an inflatable ruminant stomach. To show the relative size, a n a t o m y , a n d p o s i t i o n of the f o u r c o m p a r t m e n t s , the p r e s e r v a t i o n of a n entire r u m i n a n t stomach is a u s e f u l visual aid. The p r o c e d u r e is as follows: Soon a f t e r s l a u g h t e r remove the adipose tissue f r o m the surface. W a s h out the stomach w i t h water. R i n s e the r u m e n in W i c k e r s h e i m e r ' s F l u i d (6) : To t e n liters of boiling w a t e r add the following : g A l u m ............................................................ 333 Sodium chloride ........................................ 83 P o t a s s i u m n i t r a t e ...................................... 40 P o t a s s i u m c a r b o n a t e ................................ 200 A r s e n i c trioxide ........................................ 33
Cool a n d filter a n d to the solution o b t a i n e d above, add : liters Glycerine (or p r o p y l e n e glycol) .............. 4 M e t h y l alcohol .............................................. 1 S u b m e r g e the digestive t r a c t into the solution a n d stir daily f o r a p p r o x i m a t e l y one m o n t h . The odor will d i s a p p e a r w h e n the r u m e n is f o r all p r a c t i c a l p u r p o s e s t a n n e d a n d completely dry. A d d valves to the esophagus a n d duoden u m ends so t h a t it e s n be i n f l a t e d w i t h forced air, c a r b o n dioxide, or oxygen.

The authors express their appreciation to Fred Foltz, machinist, University of Illinois, for his help in constructing the rumen periscope. The suggestions of James' Gordon, Curator of Museum, Pathology Department, University of Washington Medical School, Seattle, Washington, and D. M. Fluharty, College of Veterinary Medicine, Washington State University, Pullman, are gratefully acknowledged.
REFERENCES

(1) BLOSSER, T. H. Improving Our Teaching in the Animal Sciences. Proc. West. Div. Am. Dairy Set. Assoc. (Pullman, Washington), 38:1957. (2) CO~BS, W. B. Use of Demonstrations in Dairy Science Teaching. J. Dairy Sci., 37: 545. 1954. (3) GORDON, ,]-. Pathology Department, University of Washington Medical School, Seattle, Washington. Unpublished data. 1954. (4~) HIx, E. L. Preparation of the Fetal Compound Stomach as an Aid in the Teaching of Ruminant Nutritional Physiology. J. Animal Set., 13:49. 1954.
(5) JONE'.S, ]~UTH MCCLUI~G. MeClung's H a n d book of Microscopical Techniques for Work-

ers in Animal and P l a n t Tissues. 3rd cd., p. 52. t t o f n e r Publishhlg Co., New York. 1961. (6) LEE, A. B. The Microtomist's Vade-Mecum. 1st ed., p. 236. Blakiston, Son and Co., Philadelphia. 1885. (7) SPALTEHOLZ, W. ~ b e r das Durchsichtigmachen yon menschlichen und tierischen PrSparten, und seine theoretischen Bedingungen, Leipzic, S. Herzel, 1911, 2 Aufl. 1914. 2 Present address: Department of Animal Sciences, Purdue University, W. Lafayette, Indiana.