J Anim Sci 1966 Johnson 855 75

Techniques and Procedures for In Vitro and In Vivo Rumen Studies Ronald R. Johnson J ANIM SCI 1966, 25:855-875.
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TECHNIQUES AND PROCEDURES FOR IN VITRO AND I N VIVO RUMEN S T U D I E S 1, 2

RONALD R . JOHNSON 3
Page
INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IN V I T R O T E C H N I Q U E S . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . :.. History . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Potential Uses of I n V i t r o Techniques . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Continuous Flow Systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Closed Systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Description of Techniques . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Buffer Solutions and Nutritional Media . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Fermentation Vessels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Degree of Agitation and Gas Phase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Temperature . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Optimum p H . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Inoculum Sources . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Fermentation Time Periods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Terminating the Fermentation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Analytical Procedures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Interpretation of I n V i t r o Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Rumen Protozoology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Pure Culture Techniques . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IN V I V O TECHNIQUES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VIVAR Techniques . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Nylon Bag Techniques . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Rate of Passage Measurements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Rumen Fistulation Techniques . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Schalk and Amadon Procedure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Jarrett Procedure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Cannula . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Other Fistulation Techniques . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Rumen Volume Determination . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Gnotobiotics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . , ....................................... SUMMARY . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . LITERATURE CITED . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
855 856 856 856 857 857 857 857 858 859 860 860 86l 862 863 863 863 864 865 865 865 865 866 867 867 868 870 870 870 871 872 872
Introduction E ruminant animal has often been classiTH fied as one which has a relatively simple dietary requirement and as such should be one which is easily studied by research techniques. Actually the contrary exists in that investigation of the metabolism and nutrition of the ruminant animal is complicated by two entirely separate metabolic systems which, although influenced by each other in some ways, can vary independently and certainly possess different metabolic characteristics. Inherently the ruminant animal generally has the same type of nutrient requirements as most other species of large animals, i.e., basic requirements for energy, protein with certain bal1 Approved for publication as Journal Article No. 82-65 by the Associate Directo~ of the Ohio Agricultural Research and Development Center, Wooster, Ohio. Paper preoared for a revision of the monograph, "Technlaues and Procedures in Animal Production Research." z Department of Animal Science, Ohio Agricultural Research and Development Center, Wooster, Ohio.
ances of amino acids, minerals, vitamins in all the classes, essential fatty acids, etc. Metabolically speaking, the greatest difference between ruminants and nonruminants is in the sources of energy upon which the adult ruminant tissues rely for a major portion of their activities. The utilization of short-chain fatty acids as immediate energy sources in the tissues of the ruminant animal, whether it be an obligatory or facultative process, has a basic effect on the animal's performance both in nature and efficiency and in addition is undoubtedly directly related to the endocrinological control of energy metabolism. With the exception of this major difference in the animal's tissues per se, the other major complicating factor in studying ruminant nutrition and metabolism is the existence of an entire metabolic system in its own right within the ruminant animal, i.e., the microbial population within the digestive tract. Although all species of large animals possess microorganisms
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JOHNSON Cuthbertson (1963), Lewis Dougherty et al. (1965). (1961) and
within their digestive tracts, only ruminants, species with an enlarged or more functional caecum, and certain marsupials can claim a major reliance on the microorganisms for their nutritional welfare. Although the metabolic system within the ruminant animal itself and the metabolic system in the microbial population definitely affect each other in many ways, they are also capable of varying almost independently in some characteristics. As a consequence refined studies of rumen microbial activity in the intact ruminant animal are subject to tremendous variations. Recognition of this fact many years ago prompted initiation of studies of the activity of rumen microorganisms by several techniques. Although those working most closely with these techniques will readily admit they leave something to be desired, it can be truthfully said these techniques have evolved to a point where they are worthy of review in a single paper for the use of investigators throughout the world. In some types of studies they can prove invaluable in studying certain biochemical phenomena associated with the rumen microorganisms. These research techniques logically can be divided into two main categories: (1) in vitro techniques and (2) in vivo techniques. The in vitro techniques depend upon complete removal of the microorganisms from the control or influence of the host animal itself, while the in vivo techniques involve either a partial control of environmental effects or possibly quantitation of the effects within the animal. Under any circumstance both types of technique are designed to elucidate the basic biochemical processes being performed by the microbial population of the ruminant and the factors which affect them. In this discussion in vitro techniques will be considered in detail first, and some discussion of various in vivo techniques will follow. It will be impossible to describe in detail all of the useful techniques that have been developed in various laboratories. By necessity the discussion will be influenced greatly by the author's personal experience, and there is no intention of leaving the implication that only the techniques described are suitable for investigational studies. For a fuller understanding of rumen microbial activity the student is referred to early reviews by Baker and Harris (1947), McNaught and Smith (1947), Phillipson (1947) and Marston (1948) and to more recent comprehensive treatments of the subject by Annison and Lewis (1959), Barnett and Reid (1961),
In Vitro T e c h n i q u e s
History
Reviews covering early developments with

in vitro investigations have been written by
Moxon and Bentley (1955) and Bentley (1959). More recently Johnson (1963) has presented a comprehensive discussion of the historical development of in vitro rumen fermentation techniques and their uses for studying rumen microbial activity. In the development and selection of an in vitro technique for studying rumen fermentation, the investigator must decide initially whether his objective is to duplicate as closely as possible the actual fermentation occurring within the rumen of the animal or merely to study qualitatively and quantitatively a few of the many processes occurring as a result of microbial activity.
Potential Uses of In Vitro Techniques
In an earlier review Johnson (1963) concluded that in vitro techniques could be utilized in the investigation of several problems: 1. Cellulose digestion and factors affecting it. 2. Utilization of nonprotein nitrogen. 3. Intermediate metabolism in both mixed and pure cultures. 4. Studies of symbiosis utilizing both all glass systems and continuous flow chemostats. 5. Studies of rate phenomena requiring a nonsteady state situation. 6. Forage evaluation studies. 7. Studies of bioenergetics of the rumen fermentation as have been investigated by Marston (1948), Hershberger and Hartsook (1960) and Walker and Forrest (1964). Undoubtedly many other uses could be added to this list; however, this range is sufficient in itself to challenge most investigators of rumen microbial activity. The main advantage of the in vitro techniques rests in the ability to use them to study activity of microorganisms away from the control and influence imposed by the host animal. Further discussion of the advantages and disadvantages will be made in the comparison of the complex
PROCEDURES FOR RUMEN STUDIES continuous flow techniques with the closed system techniques.
Continuous Flow Systems
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is its complexity and the inability to perform large numbers of experiments with it.
Closed Systems
If the attempt is to simulate actual processes within the rumen, the criteria which should be satisfied have been listed by Warner (1956) and Davey et al. (1960). The techniques and apparatus designed to satisfy these requirements are considerably more complex than those required for the other type of objective. Such systems known as "continuous flow apparatus" or "chemostats" have been designed and studied by Warner (1956), Davey et al. (1960), Gray et al. (1962), Bowie (1962), Adler et al. (1958), Harbers and Tillman (1962), Quinn (1962), Rufener et al. (1963), Stewart et al. (1961), Slyter et al. (1964) and Hobson and Smith (1963). In these techniques provision in the apparatus invariably has to be made for regular additions of nutrients in somewhat the same way as might be achieved in the actual rumen of the animal as well as for constant removal of the end products. Wolin (1960) described a theoretical rumen fermentation balance which, if systems capable of satisfying the theoretical requirements could be adopted, would enable quantitative calculations of certain microbial processes. When properly designed, conducted and interpreted, the continuous flow or chemostat techniques offer the possibility of studying rumen microbial processes as they occur in the intact rumen. Therein the processes of synthesis and absorption can be simulated and studies made of the effects of various environmental or nutrient treatments on anabolic or catabolic processes within the microbial culture. To accomplish this very exacting control must be maintained on nutrient input, end-product removal, pH, nutrient concentration, oxidation-reduction potential, rate of agitation, etc. Even with this type of control seemingly accomplished, the technique would be open to severe criticism without careful assessment of the microbial population proliferating by microscopic and bacterial counting techniques. Certainly with these degrees of control accomplished, considerable information on the microbial processes of the rumen can be obtained. However, extrapolation of the quantitative findings to the intact rumen still opens the technique to criticism, especially in the area of synthesis-absorption studies. The other chief disadvantage of this technique
In contrast, the systems designed for meeting the second objective, i.e., quantitation of a few processes occurring in the microbial population, are marked by simplicity of their design and procedure and also the ability to conduct large numbers of studies in any given series of experiments. This simplicity, however, caused the system to be subject to severe criticism and question as to whether the microorganisms being propagated were truly typical of the rumen population in the intact animal. This undoubtedly should be the concern of anyone making full use of in v i t r o techniques. As reviewed by Johnson (1963), however, a series of papers from Ohio (Dehority et al., 1960; el-Shazly et al., 1961a, b) have demonstrated quite well that bacteria propagated in vitro can be truly representative of those in the intact rumen itself. Thus, the assumption can be made that the activities being measured are similar to those occurring in the intact animal. In such a system the possibility of enriching the culture for a particular species of microorganism is always present and undoubtedly occurs to some extent in practically all closed systems. This, however, does not make the system invalid for studying certain metabolic processes, since this in fact may enhance quantitative measurements without necessarily changing them qualitatively. The possibility of culturing species with aberrant metabolic pathways should be kept in mind when using closed systems and should be checked by microbiological techniques when possible. Another major criticism of closed systems, as far as studying mixed rumen microbial cultures is concerned, is the almost invariable elimination of protozoa from the pop~Jlation. The role of protozoa in rumen microbial fermentations will simply have to be studied by other techniques.
Description of Techniques Buffer Solutions and Nutritional Media. Although many researchers have arrived at a standard buffered medium for their in vitro fermentation techniques through experimental studies, most of the media are based on socalled "McDougatl's solution", which is an artificial saliva or buffer medium based on the
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TABLE 2. C O M P O S I T I O N O F " O H I O " FERMENTATION MEDIA Ingredient Na2CO3, 200 m g . / m l . Mineral mixture ~ F e C h , 4.4 m g . / m l . C a C h , 5.29 m g . / m l . U r e a , 126 m g . / m l . B i o t i n , b I0 m c g . / m l . Valeric acid, b 5 mg./ml.
analysis of sheep saliva by McDougall (1949). Many of the procedures specify that this solution alone is the buffer medium used in their in vitro fermentation studies. Others have added modifications for particular reasons. Burroughs et al. (1950a, b) in one of the earlier in vitro fermentation studies used McDougall's solution modified by the addition of a series of trace minerals. Some workers such as Donefer et al. (1960) have increased the buffering capacity of the solution, in order to eliminate the necessity of adjusting p H in the middle of a fermentation run. Others have varied the proportions of phosphates and bicarbonates as part of the buffering system. Anderson et al. (1956) and Hall et al. (1961) used an in vitro rumen fermentation system for studying the availability and utilization of phosphorus in various phosphorus supplements by eliminating phosphates from the buffering medium. Since the discovery of the requirements for certain short-chain fatty acids and biotin as growth factors for cellulolytic rumen microorganisms by Bentley et al. (1954a, b, 1955) and Bryant and Doetsch (1955), valeric acid and biotin have become standard ingredients of the in vitro fermentation media used in our laboratory when cellulose digestion is the variable being measured. Both nutrients are especially critical when separated cells or washed inocula are being used. A number of other laboratories have adopted this procedure. The composition of McDougall's solution is shown in table 1. The medium routinely used in our laboratory for in vitro rumen fermentations is shown in table 2. Variations of these media will obviously be required depending on the types of activity being studied. For example, the form of nonprotein nitrogen supplied to support microbial growth may be varied in any number of ways. Cellulolytic bacteria are considerably more sensitive to high levels of urea nitrogen than are starch-digesting bacteria, i.e., higher levels
TABLE 1. C O M P O S I T I O N OF ARTIFICIAL SALIVA a G m . / l i t e r of distilled H~O
I N VITRO
M 1 . / 1 0 0 ml. 1.0 2.0 1.0 1.0 1.0 2.0 5.0
a The mineral mixture consists of the following dissolved and diluted to 1 liter in water: Na~HPO4, 56.5 gin.: NaH~PO4, 54.5 gin.: KCI, 21.5 gm.; NaCl, 21.5 gm.; MgSO~'7 H20, 5.82 gm.; and K2SO~, 7.50 gin. b Biotin and valeric acid have been shown to be essential only for cellulolytie rumen bacteria, but are added routinely in this laboratory in most media.
Ingredient
NaHCO3 KC1 CaCI~ Na2HPO4" 12 H~O NaC1 MgSO4" 7 H_~O

a McDougall (1949).
9.80 0.57 0.04 9.30 0.47 0.12
of urea nitrogen are toxic to cellulolytic activity in mixed rumen cultures. At least two to three times as much urea per unit volume of medium is required to be toxic to starchdigesting activity. Yet the supply of nitrogen can be a critical factor, especially when both starch- and cellulose-type substances are being digested in the same fermentation (elShazly et al., 1961a). When washed cell inocula are being used, responses to some trace minerals and other growth factors have also been shown. However, if a significant quantity of rumen fluid is carried with the inoculum or is added to the medium, most of the trace growth factor requirements are supplied therein. Fermentation Vessels. The apparatus for the continuous flow or chemostat type in vitro rumen fermentations will not be described in this paper, and the reader is referred to the references cited. For the closed-system type fermentation, practically any type of vessel may be used (el-Shazly et al., 1960). Two typical systems are shown in figure 1. Generally speaking all-glass systems have been used by most workers in recent years. In our laboratory fermentation vessels varying from 10 to 1,000 ml. in fluid volume have been utilized, although in vitro fermentations up to 360 liters have been used elsewhere (Hershberger and Hartsook, 1960). For studies with fibrous substances such as cellulose or forages, it is most convenient to utilize a vessel and system which wilt involve the least possible number of transfers of fiber-containing media prior to final analysis. For example, the substrate in question may be weighed directly into a centrifuge tube which is also used as the fermentation vessel. At the end of the fermentation time the tube can be centrifuged and the supernatant discarded. Further analy-
P R O C E D U R E S FOR R U M E N STUDIES
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$
-'~--SLIT
BUNSEN VALVE IN VITRO

FERMENTATION TUBE Figure I. (A) In vitro fermentation tube showing stopper for gassing with CO~ and (B) Bunsen valve showing outlet slit for escape of gases.
ses can be conducted on the residue in the same tube, thus avoiding any transfers whatsoever between the initial weighing and the actual chemical analysis. If it is desirable to obtain samples at different times from the same fermentation, however, larger volumes will often be desired. In this case it is usually convenient to use a large-mouth bottle which has been calibrated for the appropriate volumes. Samples may be removed from these bottles by rapid agitation either by a mechanical stirrer or by hand, while a sample is being removed with a pipette with a large opening to permit passage of fibrous particles. To avoid excessive contact with oxygen during this sample removal it is convenient to prepare a rubber stopper with a gassing tube extending to the bottom of a fermentation flask or bottle. A large V-slot cut in the edge of the rubber stopper permits escape of the carbon dioxide that is used to rapidly gas the fermentation media during sampling and also permits the entry of a pipette into the medium. Violent gassing together with rapid agitation by hand permits a uniform sample to be taken. A problem arises when a series of samples are to be taken from the same fermentation in that some evaporation of the liquid volume occurs between samplings. Hence, calibration on the bottles is necessary to facilitate dilution of the fermentation medium back to the
proper volume. For example, D ehority (1961) and Dehority and Johnson (1961) removed samples at different times from the same 175ml. fermentation bottle by calibrating the bottle at 100, 125, 145, 160 and 175 ml. Thus, samples of 15, 15, 20, 25 and 25 ml. could be taken at successive times during fermentation. The larger samples in the later time periods were found to be necessary, since much of the cellulose had disappeared and more volume was needed for accurate analysis by the chemical method. Serial time samples also may be taken by using replicate fermentations in separate vessels. However, in choosing this technique one must weigh the variability between replicates against the pipetting error inherent in the first method. Either method described is equally appropriate for studying other substrates as well. For example, the study of digestion of other carbohydrate substrates may be somewhat simpler, since a nonfibrous type of substrate is used. Nevertheless, when starches are being studied, severe clumping during certain periods of the fermentation sometimes occurs and makes homogeneous sampling difficult. In vitro techniques have also been used to study the utilization of various nonprotein nitrogen compounds and some mineral compounds by rumen bacteria. The main caution in this case is presented in the case of materials which are relatively insoluble. In such a case they usually settle to the bottom of the fermentation vessel and are not readily sampled b y a pipetring technique. For this reason the sample taken at zero time and at a later time may not be representative of the same continuous fermentation. This can be readily solved by using individual fermentations for each time sample. Degree o/Agitation and Gas Phase. In vitro rumen fermentations have been conducted both with and without mechanical agitation. It is the opinion in many laboratories that agitation, even with insoluble substrates, is not generally necessary. Unpublished results from our laboratory indicate that violent agitation is actually deleterious to the digestion of cellulose. I t has been common practice in most techniques to insure a complete mixing at the time of inoculation and at the time material is added to or removed from the flask. If the flask is continuously gassed with carbon dioxide, this also provides a mild movement within the liquid medium of the fermentation vessel. Two main types of gassing systems have been used, both of which recognize the strict requirement for anaerobiosis for
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JOHNSON to them for significant periods of time, a loss in activity is often noticed. Optimum ptt. Since the buffering capacity of the medium itself is the only means by which p H can be controlled internally in an all-glass system, the p H in these systems often changes during the process of fermentation. I t is quite likely that this will have to be adjusted. The p H optimum for cellulose digestion has generally been found to be around 6.9. Moore et al. (1962) found that the p H optimum for starch digestion was 6.8. Undoubtedly, the p H optimum for some of the other activities carried on by rumen microorganisms may vary from these two points. Nevertheless, it can be safely said that most of the. rumen microbial activity occurs at an optimum rate between a pH range of 6.7 and 7.0. The necessity for adjusting p H depends largely upon the rate, extent and type of fermentation. During the course of cellulose digestion acid production occurs at a much slower rate than during starch digestion. As a consequence, when the buffering medium described earlier is used, the p H needs to be adjusted only two or three times during a 24hr. fermentation period when cellulose is the main substrate. If starch digestion is being studied, however, the p H may drop much more rapidly due to the rapid rate of fermentation of this more soluble type of carbohydrate. In studies on adapting all-glass fermentation systems to starch digestion, Moore et al. (1962) found that the p H did not drop markedly until between 6 and 12 hr. after inoculation. After the fermentation started, however, the p H dropped very rapidly, and for maximum activity it was necessary hourly to readjust the p H to 6.8. A stronger buffer medium, of course, may have alleviated part of this problem. If soluble carbohydrates such as glucose are being used as substrates, p H changes may commence during the first hour after inoculation, and the drop in p H may be much more rapid. When a rapidly hydrolyzed source of ammonia nitrogen such as urea is being used as a nitrogen source, the effect of acid production on pH is modified. In some cases where ammonia production is very rapid and acid production has not commenced, the p H may actually rise above 7. This is usually quite temporary but may be deleterious, since the rumen bacteria seem to be somewhat more sensitive to p H levels above than below 7. Generally, p H is adjusted from the acid side to 6.9 with a solution of sodium carbonate
the proper culture of rumen bacteria. The first technique is a continuous gassing in which CO2 is bubbled slowly through the medium by placing a gassing tube near the bottom of the fermentation vessel. The second technique provides for an initial thorough saturation of the medium with CO z followed by closing the fermentation vessel with a stopper containing a Bunsen valve (figure 1). The latter item is usually no more than an inverted rubber policeman placed over an outlet tube. A small slit cut in the rubber of the policeman allows gas to escape, but air is prevented from entering the system. Both techniques have been used quite successfully in various laboratories, and the decision as to which is preferable should be determined experimentally by the operator. The continuous gassing technique is probably more advisable if several serial samples are to be taken at intervals during fermentation, since it insures the maintenance of a CO2 atmosphere at all times. However, this technique has the disadvantage of a somewhat more complicated apparatus, since it is most successfully performed with individual tubes leading from a common manifold to each fermentation vessel. The Bunsenvalve technique depends on the ability of the fermentation to produce enough CO2 during the phases of metabolism to keep the atmosphere and the liquid medium saturated. In most fermentations the CO2 produced would be more than adequate for this purpose as long as no air is permitted to enter the flask. However, if serial samples are to be taken, the vessel needs to be resaturated with CO2 by fairly violent bubbling after each sampling. This violent agitation may have an effect on the fermentation for a short period of time after agitation. Temperature. The temperature usually selected for in vitro fermentations has been 39 ~ C., although occasionally others are reported. Although constant temperature water baths are generally preferred as the means of maintaining temperature, some investigators have used incubators for this purpose. Either method is satisfactory as long as a reasonably constant temperature is maintained. However, temperature should definitely be standardized, since differences of as little as 0.5 ~ C. may invalidate comparisons between individual fermentations. In addition, care should be taken to prevent the temperature from rising over 40 ~ C. during all phases of the fermentation. Rumen bacteria appear to be especially sensitive to high temperatures and, if exposed
P R O C E D U R E S FOR R U M E N STUDIES (saturated) and adjusted from the basic side to 6.9 with a phosphoric acid solution. Other acidic and basic solutions could possibly be used to adjust the pH; however, these two correspond to the usual buffering system employed in the media. Inoculum Sources. The source of inoculum for in vitro rumen fermentations also represents the source of the greatest possible error or variability as well as the possibility for misinterpretation of the results. The two major considerations are (a) the choice of the source animal or animals and (b) the methods of inoculum preparation. These will be considered in the order given. Several investigators (Warner, 1956; Bowie, 1962) have pointed out the importance of utilizing a source animal, which is being maintained on a ration similar to that being studied in vitro. This is especially true for those using the continuous flow or chemostat techniques. Although the rumen has the advantage of abounding in a variety of species and types of bacteria and protozoa, it also has the concomitant disadvantage of the ability of these groups of microorganisms to change in proportion to each other very rapidly. Many times these changes are due to the substrate or, in the case of the animal itself, to the ration being fed. Although it might seem convenient to classify rumen microorganisms by substrate-fermenting activity such as cellulolytic, amylolytic, glucose digesters, proteolytic, etc., it is not technically possible to do this since many of the groups of bacteria overlap in classification. Nevertheless, it stands to reason that, if one is studying starch digestion, the inoculum should be taken from animals that are fed a ration which has a predominance of starch as the carbohydrate substrate. The same rule is true for studies of cellulose digestion and other investigations. In recent years numerous investigators have studied the use of in vitro fermentations for the evaluation of forages. Most of these workers have found it convenient to standardize one or more animals on a standard roughage feed for the entire year or more and by so doing have found that the variability of the inoculum from day to day from such animals can be minimized by careful procedures during preparation of the inoculum. This is especially important, when one is attempting to compare quantitatively the data for more than one day. In the study of starch digestion Moore et al. (1962) found it convenient and advisable to use four inoculum-source animals
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on the same ration and composite the inoculum drawn from all four animals on any given day of study. By so doing the chance of one animal's being slightly "off feed" and thus grossly affecting the in vitro results would be minimized. I t has been our experience that it is easier to detect an animal that is "off feed" by studying its inoculum in the laboratory than by observing the animal in the feeding pen. When determining forage digestion in vitro some workers have observed that inocula taken from animals fed alfalfa hay were superior to inocula taken from animals fed grass hay. Although no explanation can be offered for this effect, it emphasizes the importance of considering the source of inoculum, even when the variable under study is merely the type of forage. Methods of inoculum preparation vary from the simplest technique of using whole rumen fluid taken directly from the animal's rumen to the use of washed cell suspensions and enrichment cultures. Again the choice of preparation techniques depends on the phase of rumen activity being studied. Whole rumen fluid may be removed from the rumen by two basic techniques. The first is by aspirating or drawing off a sample of rumen fluid directly from the rumen by vacuum techniques. Usually a plastic tube with a series of inlet holes drilled in one end is used to sample several sections of the rumen to obtain a random sample from the various compartments. The other technique is simply to remove a sufficient quantity of rumen contents by hand, place it in a press and squeeze the liquid from the material. This type of inoculum is invariably filtered through several layers of cheesecloth. I t should be kept in mind that when this type of inoculum is being used it carries with it the mother liquor, which contains a host of nutrients and growth factors for the microorganisms. If the object of the study is to measure the requirement for some of these nutrients, then it would be advisable to eliminate this mother liquor. On the other hand, if the object is to obtain maximum activity, it may be desirable to retain this liquid. I t also represents the fastest means of transferring microorganisms from the rumen to the in vitro fermentation flasks. A modification of this technique was developed by Johnson et al. (1958), in which the rumen contents were squeezed in a large press and the first liquid extract was discarded. The squeezed pulp was then resus-
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et al. (1960) by growing cellulolytic bacteria in an in vitro system for a period of time and
pended in a pH 7 phosphate buffer, agitated and re-squeezed. This second extract was then used as the inoculum and has become known as the "phosphate buffer extract." This procedure was developed with the fact in mind that many of the cellulolytic organisms exist in close association with the fibrous portions of the tureen contents as demonstrated by Baker and Harris (1947). By using this inoculum in cellulose digestion studies, both increased digestion and improved precision were obtained. This technique would not necessarily be advisable when one is studying digestion of starch or other ration constituents, however. More refined inoculum preparation techniques depend usually on various means of centrifuging and washing the tureen bacterial preparation with a buffer mixture. Bacterial cells separated from whole rumen fluid by high speed centrifugation were first employed by McNaught (1951) and later to a greater extent by Bentley et al. (1954a, b, 1955). The latter workers found it feasible to use a Sharples supercentrifuge to concentrate the bacteria in batch form to prepare the inoculure. Although the inoculum developed by Johnson et al. (1958) was almost completely inactivated by two washings with phosphate buffer, Cheng et al. (1955) developed a procedure using washed cell suspensions, which was used to study nutrient requirements of tureen microorganisms by Hall et al. (1953) and Hubbert et al. (1958a, b). Since then several workers have used washed cell suspensions successfully in in vitro rumen fermentations. Dehority et al. (1960) purified the cellulolytic inoculum even further by separating the fraction sedimented between 1,500 and 3,000 g. In studying starch digestion Moore et al. (1962) centrifuged the tureen bacteria at 3,000 g and resuspended this sediment for an inoculum. In the preparation of any washed cell suspension or centrifuged inoculum, the potential inactivation of the microbial activity by both exposure to oxygen and adverse temperatures must be kept in mind. The gradual but steady loss in cellulolytic activity by incubation and aeration was demonstrated by Johnson et al. (1958). Nevertheless, these techniques may be necessary in order to free the bacterial preparation of endogenous nutrients and growth factors. For example, a more exact requirement for short-chain fatty acids by microoganisms was demonstrated by Dehority
then reharvesting the cells as an inoculum for future fermentations. By so doing blanks giving zero cellulose digestion were obtained. Fermentation T i m e Periods. The time of fermentation for in vitro tureen studies is entirely the function of the objectives of the study itself. Time periods from several hours to several days have been used for various studies in the past. A few basic comments may enable the student to select a time period more intelligently. An obvious wide variation exists in the times required for both initiation of fermentation and maximum rate of digestion of various types of carbohydrates and other substrates by rumen microorganisms. Soluble carbohydrates such as simple sugars are readily fermented within a matter of minutes after combining with the inoculum and often peak activity occurs within 1 to 2 hr. Initiation of starch digestion may require a longer period of time, depending upon the type of inoculum being used. However, after starch fermentation has started it proceeds very rapidly, and in most fermentations there is rarely little starch left for fermentation after 24 hr. (Moore et al., 1962; el-Shazly et al., 1961a). Highly soluble starches will be more quickly fermented than the less soluble forms. On the other hand cellulose digestion not only is initiated after a longer period of incubation, but also usually proceeds at a slower rate than digestion of more soluble carbohydrates. In many in vitro systems, however, a distinct difference exists between the digestion of cellulose in its native form in forages and the digestion of purified cellulose such as Solka Floc. 4 Digestion of native forage celluloses appears to be initiated at a much earlier time, i.e., digestion is detectable after 6 hr. of fermentation, whereas the digestion of purified cellulose often is not initiated until after 12 hr. or more of fermentation. The exact reasons for these differences are not known as yet. Baker et al. (i959) have shown that the rate of digestion of purified cellulose decreases with increasing degrees of crystallinity. They further suggest that the native cellulose in forages is mostly
9 The Solka Floc mentioned herein is a product of the Brown Paper Company, Berlin, New Hampshire, and is mentioned only because it has been accepted by most workers in the rumen field as a standard cellulose source for in vitro tureen fermentations. I t should be kept in mind, however, that there are several grades a4~d types of Solka Floc produced by this company; the one to which we refer in this paper is Solka Floc BW-40.
P R O C E D U R E S FOR R U M E N STUDIES in the amorphous form, which may partially explain the difference in the rate of digestion. At any rate, if a purified cellulose is being used as a standard, some knowledge of its physical properties is desirable. A further consideration should be made when the objective involves measurement of rate phenomena. In most closed systems it is impossible to provide sufficient substrate to maintain the maximum rate of fermentation for long periods. In other words the substrate usually becomes limiting soon after the maximum rate of digestion has been achieved. This should be considered in selecting the times for measurement of rate processes, and preliminary work involving studies on substrate level as it affects rate and extent of digestion is mandatory. Terminating the Fermentation. Practically any means of stopping enzymatic activity can be used to terminate in vitro rumen fermentations. In some cases it suffices merely to remove the tubes from a water bath and refrigerate them. For more exact timing of the termination of fermentation, additions of acids, alcohol and mercuric chloride are often practiced. The addition of acids has the disadvantage of causing the evolution of considerable CO2, which may create sufficient foaming to coat the sides of a vessel with portions of the substrate material. Assuming it does not interfere with subsequent analytical procedures, the method of choice used in our laboratory is the addition of approximately 1 ml. of saturated mercuric chloride solution per 100 ml. of fermentation volume. Analytical Procedures. No attempt will be made to elucidate the numerous analytical procedures that might be used in following the activity of in vitro rumen fermentations. For most purposes procedures that are useful for the particular materials being studied can be modified to be suitable for analyzing in vitro rumen fermentations. I t must be kept in mind in interpreting results, however, that while substrate may be disappearing during the fermentation, a large quantity of bacterial cells is being produced--a fact which may complicate the terminal analysis. For example, studies on the proteolysis of natural feed proteins have had to be restricted to indirect measurements of proteolysis such as ammonia formation, since there is no easy way to distinguish between the natural feed protein and the microbial protein being synthesized. Interpretation of In Vitro Results
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In a discussion of the interpretation of in vitro rumen fermentation data, one can only enumerate points of caution that should be considered. Actual methods of interpretation or analysis of data must be left to the individual investigator. Firstly, the author chooses to reiterate an objection to the term "artificial rumen," since the closed systems used are generally in no way intended to duplicate exactly what is happening in the rumen. The term "in vitro rumen fermentation" is preferred. Criteria for validity for such fermentations have been published by Warner (1956) and Davey et al. (1960). In the design of any system, be it a closed system or continuous flow system, it would be desirable to satisfy as many of these criteria of validity as possible. Nevertheless, closed system procedures are often usable for studying specific phases of rumen microbial metabolism, and in doing so one or more criteria may have to be sacrificed to accomplish the task. Consequently, the criteria which have been sacrificed must be kept in mind when interpreting the results. I t is one thing to demonstrate a fact in the closed in vitro fermentation system and still another to show that similar processes occur in the intact rumen of the animal. Therefore, transposition of conclusions is not necessarily justifiable in all cases. Much of the data obtained to date with in vitro fermentations have been obtained using purified media, which greatly simplify the techniques and analytical procedures involved. At the same time, however, this automatically prevents the system from being compared directly with natural animal rations and the processes within the rumen. The chemical nature of natural feedstuffs is indeed a complex one, and the digestion of the individual chemical constituents of these feeds is a subject which needs to be studied a great deal in the future. I t is of immediate interest that the digestion of forage substrates has been studied to a considerable extent recently by use of in vitro rumen fermentation techniques. Many workers have been engaged in both independent and collaborative studies in the use of in vitro rumen fermentations as a means of studying and evaluating natural forages as feedstuffs for ruminants (Pigden and Bell, 1955; Kamstra et al., 1958; Quicke et al., 1959; Hershberger et al., 1959; Donefer et al., 1960; Baumgardt et al., 1962a, b, 1964; Reid et al., 1964;
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JOHNSON to this variation, if each of the laboratories were calculating regression equations from their in vitro data and were using standard in vivo data, widely different regression lines would be obtained. In addition to this, a collaborative in vivo digestion trial (E. Donefer, unpublished data, MacDonald College, Quebec, Canada) demonstrated that considerable variation occurred also in the conduct and results of in vivo trials, even when a standard forage was used. These reports simply point out that, with the techniques presently in existence, it would not be possible for one laboratory to use the regression equations developed in other laboratories, even though identical techniques may be in use in both laboratories. It has appeared to be convenient and popular, especially in some other countries, to utilize in vitro rumen fermentations to study a series of forages and their digestibility without accompanying in vivo digestion trials to establish proper regression equations. Indeed, this is the ultimate goal of those working with the techniques. However, the validity of the regression equations should be established by at least a limited amount of in vivo work prior to this type of comparison. Certainly this is true when wide variations in species of forages are being investigated. For example, it would not seem advisable to use regression equations developed for temperate-region forages when one is studying tropical or semitropical grasses. The in vitro system by itself may serve the purpose of classifying forages in relation to one another as far as digestibility is concerned, however. Once a technique has been standardized and regression equations have been determined, the in vitro rumen fermentation may prove highly valuable in studying forages. Generally, data can be obtained by many of the techniques which are highly correlated with dry matter digestibility. This has been readily shown by Baumgardt et al. (1962a, b, 1964), Johnson et al. (1964), Hershberger et al. (1959), Reid et al. (1964) and Tilley et al. (1960). An excellent application of these techniques to the measurement of herbage digestibility and intake by grazing livestock has been published by VanDyne (1963), VanDyne and Meyer (1964) and VanDyne and Weir (1964).
R u m e n Protozoology
Johnson et al., 1962a, b, 1964; Tilley et al., 1960; Tilley and Terry, 1963; Barnes, 1966). The general procedures used in most of these studies have been outlined in preceding sections and will not be repeated here. For exact procedures the reader should refer to the literature. On the other hand there does seem to be some inconsistency in the interpretation of this type of information. Because of the interest shown by workers in many other countries in these particular techniques, some discussion of this interpretation will be given here. The usual procedure for developing in vitro rumen fermentation techniques to study and evaluate forages for ruminant animals has been to take a selected group of forages and feed them to animals in such a way as to obtain digestibility and intake figures. The same forages are used for in vitro rumen fermentation studies, and the results from the in vitro studies have been correlated by regression techniques with in vivo data from the animal trials. Regression lines are established which provide a means of predicting the nutritive value of other forages studied by the in vitro technique. This is possible because the characteristics of forages differ greatly not only among species but also within species due to factors such as stage of maturity and harvesting techniques. One very important fact has emerged from these studies which deals with the interpretation of the data. Many species of forages form a uniform pattern within species in their relationship between in vitro and in vivo data. Indeed some species can be grouped together and still form highly significant patterns. Nevertheless, definite and gross differences among some species have been observed and have necessitated the use of considerable caution in interpreting results. For example, Johnson et al. (1962b) observed that a difference existed between alfalfa and grasses in the rate at which their digestibilities decreased due to stage of maturity. This suggests that, for most exact interpretation of such data, regression lines should be available for each species of forage tested. To further complicate this consideration a collaborative study has recently been completed in which 17 laboratories used various in vitro fermentation techniques to study the digestibility of standard forages (Barnes, 1966). This study demonstrated that considerable variability occurred within and between the techniques employed at the different laboratories. Due
When the large populations of protozoa
P R O C E D U R E S FOR R U M E N STUDIES possible in rumen fermentations are considered, it is unfortunate that their role in overall metabolism has not been more fully studied. In surveying the literature on this subject, the student will soon realize that one of the reasons for this paucity of information is the difficulty encountered in culturing protozoa in vitro. Although several workers have succeeded in keeping rumen protozoa alive for several weeks in vitro, to the best of the author's knowledge none of these preparations has been completely free of bacteria. The close association of some protozoa and rumen bacteria thus makes interpretation of metabolic data very difficult. No review of the techniques for study of rumen protozoa will be given here, but the reader is referred to the following references (Clarke, 1963; Coleman, 1960, 1962; Eadie, 1962a, b; Gutierrez, 1955; Guderrez and Davis, 1959, 1962; Hungate, 1955; Hungate et al., 1964; Mall, 1964; Mah and Hungate, 1965; Oxford, 1955; Quinn et al., 1962; Warner, 1962). Pure Culture Techniques The study of rumen microorganisms in pure culture is ultimately necessary if the actual pathways of metabolism are to be studied thoroughly. Techniques for this phase of rumen investigations, although demanding, have been developed and are in use throughout the world. Discussion of the techniques, however, is beyond the scope of this review and would be a subject for a complete review by itself. For further detail the reader is referred to Bryant (1959), Hungate (1950) and Hungate et al. (1964). In Vivo Techniques Although no attempt will be made in this review to give a complete coverage of in vivo techniques for investigating rumen function, it is felt that some mention should be made of a number of them, since they bear directly on the studies of the function of rumen microorganisms. The discussion will be restricted to techniques other than digestion trial techniques, since these are discussed in ~nother chapter of this monograph. V I V A R Techniques The VIVAR technique refers specifically to an "in vivo" artificial rumen apparatus originally described by Fina et al. (1958) and later modified by Teresa (1959) and Fina et
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al. (1962). Basically, the apparatus consists of either a stainless steel device or a glass jar in which a rumen fermentation is conducted, while the unit is suspended in the tureen of a fistulated animal. The fermentation within the apparatus is separated from the environment of the rumen by a bacteriological membrane. The object of this device is to enable the investigator to study rumen microbial activity in a semiclosed system, yet one which is in equilibrium with the environment of the rumen itself. Conditions and times of equilibrium of solutes between the two phases can be modified by selection of bacterial membranes or filters of different porosity. The inner contents of the apparatus require the provision of a medium, substrate and inoculum in the same manner as an in vitro fermentation. Pettyjohn et al. (1964) described a different type of apparatus which works on essentially the same principle in studies with digestibility of forages. In their apparatus small dialyzing sacks inoculated with rumen liquor were placed in perforated plastic cylinders, and the cylinders were then placed in the rumen. Nylon Bag Techniques A number of investigators have studied the digestion of forages in the rumen by the use of the nylon or dacron bag technique. In this technique bags made of an indigestible material such as dacron or nylon are filled with the substrate in question, usually forages, and tightly tied (figure 2). These bags are then placed in the tureen of a fistulated animal by a variety of techniques and removed after various periods of time to determine digestion of the contents. Precautions must be taken to insure a mesh sufficiently fine that particles of the test substrate cannot pass through the material and in addition to insure that there are no holes in the bag. Most workers made provisions for suspending the bags in the rnmen in such a way that they would not lodge in the bottom or in any particular pocket of the rumen. Hopson et al. (1963) compared this technique with determination of digestibility by in vitro procedures. Generally, the coefficients of variation for the dacron bag technique were very high for digestibility values at early time periods from 6 to 24 hr. The digestion curves obtained from using these techniques, however, were similar to digestion curves obtained using in vitro fermentations. I t was also note-
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R a t e o/ Passage M e a s u r e m e n t s
Figure 2. Nylon bag shown with nylon suspension cord and identifying tag.
worthy that an effect of the ration of the animal on digestibility of the substrates within the bags was noted. Although the variability and the values from such techniques appear to be high, the dacron bag technique as well as the VIVAR technique appears to have some application in determining the effect of various ration treatments on digestion within the rumen. I t also represents one of the few methods available to study rate phenomena in the rumen of the intact animal. For further details see el-Shazly et al. (1961a), VanKeuren and Heinemann (1962), Burton et al. (1964), Belasco et al. (1958), Erwin and Elliston (1959), Miles (1951), Quin et al. (1938), McAnally (1942, 1943), Balch and Johnson (1950), Archibald et al. (1961) and Lusk et al. (1962). Although similar in principle, a considerably different technique mechanically is the use of suspended cotton threads in the rumen, (Hoflund et al., 1948; Balch and Johnson, 1950; Campling et al., 1961). This technique has been used to study rumen microbial activity involving cellulose digestion. In this case the threads are weighed and suspended in the rumen for a period of time, after which they are withdrawn and washed thoroughly. After drying they are weighed again and the activity is determined by loss of weight. Australian workers have recently shown that analysis of the coils of thread for nitrogen after periods of suspension in the rumen yields data which also correlate well with microbial activity (R. J. Moir, personal communication).
Although not a direct measure of rumen microbial activity, rate of passage of food residues through the digestive tract of the ruminant has been accorded importance in recent years due to the increased knowledge of the process of rumen digestion, especially in reference to forages. Since a considerable portion of ruminant rations may be indigestible, in some investigations rate of passage could conceivably be a more important measurement than digestibility. Reid (1961) has estimated that 90% of the variation in total energy intake between forages can be accounted for by variations in the intake. Voluntary intake of the forages is in turn related to rate of passage (Blaxter, 1962). Rate of passage through the entire .digestive tract has been most conveniently measured by those techniques described by Balch (1950) and Castle (1956), in which portions of the ration are stained with a particular dye and the number of stained particles excreted in the feces over a period of time are counted visually. Equations for calculation of mean retention time were described by Castle (1956). In the determination a cumulative excretion curve is drawn, and the times for excretion of 5% to 95% (at 10% intervals) of the total particles excreted are determined from the curve. These times are summed and divided by 10 to determine " R " or mean retention time. These techniques are extremely laborious and at best are only estimates of rate of passage. Care must be taken to insure that the stained material resembles physically the component of the ration that is being studied. A modification of the use of stained feed particles is the use of inert plastic particles. These can be manufactured with different size, color and density and thus present an array of characteristics which may be tested. King and Moore (1958) compared the passage of plastic particles of different size and density. Although the use of stained particles is recommended when rations of different texture or digestibility are studied, it is conceivable that measurements of rate of passage of rations with similar texture and digestibility could be made by simpler indicator techniques. Such a technique was employed in our laboratory (unpublished data). The rate of passage of both roughage and concentrate rations (but with all animals getting the same ration dur-
P R O C E D U R E S FOR R U M E N STUDIES ing the same period) was studied by dosing the animals with chromic oxide in capsules. Feces were collected at various intervals over a period of time and then analyzed for chromic oxide. The excretion pattern of the chromic oxide could be plotted against time in the same manner as the excretion of stained particles, and a mean retention time could be calculated. Powdered polyethylene has also been used as an indicator in rumen studies (Chandler et al., 1964). I t is the opinion of the author that considerably more work should be conducted on techniques for measuring rates of passage of feed residues in ruminant animals.
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Rumen Fistulation Techniques Although the presence of several distinct anatomical portions in the digestive tract of the ruminant may be beneficial as far as the animal's welfare is concerned, it complicates the study of the digestive process and the rate of feed passage through the digestive tract. In recent years surgical techniques have been developed and refined to the point that direct access to practically any portion of the digestive tract is possible. I t is further possible not only to obtain samples from these various portions of the digestive tract, but also to recycle the contents back into the tract after sampling or after making certain additions at specified points along the tract, These techniques should aid greatly in the study of the digestive process in the ruminant, especially that process which occurs beyond the reticulo-ruminal area. Rumen fistulation techniques were described many years ago by Schalk and Amadon (1928), and since that time numerous modifications of the fistulation techniques and design of cannulas have been developed. The procedure described by Schalk and Amadon (1928) was essentially a one-stage surgical technique, while the more recent procedure described by Jarrett (1948) consists of a twostage operation. Both techniques have been widely used. Since the original publications by Schalk and Amadon (1928) and Jarrett (1948) may not be readily available to the student desiring to know more about fistulation procedures, a more detailed description of these procedures will be given. The descriptions will include modifications and techniques used in the author's laboratory and do not necessarily reflect the exact description published b y the original authors. Schalk and Amadon Procedure (Figure 3).
The animal should be fasted for 24 hr. before the operation. Just prior to the operation it has been found advisable to treat the animals with a general tranquilizer. Both the Schalk and Amadon and the Jarrett operations may be performed on an animal in the standing position, although the original Jarrett procedure was described for a fully-anesthetized animal lying on its side. After the animal has been tranquilized, the left flank region between the last rib and the hip bone is dipped, shaved, scrubbed with an antiseptic soap and painted with a tincture of iodine solution. The line of incision is anesthetized locally with a 2% procaine solution by injecting subcutaneously and intradermally. Some workers have recommended that 0.002% adrenalin be included in the procaine solution to promote vasoconstriction in the incision area. After the area is sufficiently anesthetized, a vertical incision is made extending from a point just ventral to the transverse lumbar processes downward for a distance which is determined by the size of the cannula to be inserted in the animal. The skin, fascia and muscle tissue are incised or dissected b y blunt dissection techniques. After picking up the peritoneum on either end of the operative area with hemostats, it is cut and the rumen wall is exposed. A fold of the rumen wall is grasped and pulled out through the incision in the abdominal walI. The dimensions of the fold are determined by the size of the incision and the size of the cannula to be inserted. With the wooden clamp opened as far as possible without removing the nuts, the fold in the rumen wall is drawn through the clamp, which is then closed by turning the nuts. I t is important to insure that the clamp is tight and compression is sufficient to completely cut off the Mood supply of the isolated fold of rumen tissue. The rumen wall is then sutured to the skin, just below the clamp, with as many sutures as seem desirable for the length of incision involved. The area of the wound may then be treated topically with antibiotic materials and sprayed with plastic bandage. The sutures may be removed after 7 to 9 days, at which time the necrotic section of the rumen wall may be excised and the clamps removed. I t may be desirable merely to wait until the necrotic section has dropped away prior to subsequent treatment. I t may also be necessary at that time to treat minor lesions still remaining along the incision line prior to inserting the cannula in the fistula. As soon as the swelling has subsided and healing is
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Figure 3. Several stages of Schalk and Amadon (1928) fistulation procedure: (A) incision with rumen fold pulled through, (B) placing wooden clamp on rumen fold, (C) inserting sutures along clamped area, and (D) tying sutures.
nearly complete, a cannula should be inserted to close off the rumen. I t is impossible to obtain a fit sufficient to prevent leakage of rumen fluid with this procedure, especially with larger openings. However, some experience must be gained by the operator before the proper selection of the size of incision and rumen fold can be made to allow for the desired fistula opening. Jarrett Procedure (Figure 4). For this procedure the animal is similarly fasted and tranquilized. The area of surgery is cleaned and anesthetized locally with procaine solution. The surgical incision is made midway between the last rib and the hip joint, starting approximately 2.5 cm. below the transverse processes. It is continued ventrally for about 7 to 10 cm. for sheep destined to be fitted with the original Jarrett cannula. The two edges of the incision are folded back by the attachment of hemostats placed along the edge. The superficial fascia which lies immediately below is
divided by blunt dissection. If there is a heavy fat deposit, it may be more convenient to remove some of the fatty tissue. Blunt dissection techniques are used to divide the three distinct layers of muscle fibers lying between the surface and the peritoneum. After the initial dissection, the muscle layers are then separated by means of the fingers to give access to the peritoneum. When the peritoneum is reached, it should be incised in such a way that the two edges of the incision can be picked up and held with hemostats. This incision should be made about 2.5 to 3.5 cm. long in the same direction as the original incision. A pocket of the rumen is drawn out through the incision and held by means of lightly tensioned forceps until the first few stay sutures have been inserted. Four or five stay sutures are inserted around the base of the exteriorized rumen pocket by picking up the muscular wall of the rumen with the suturing needle, continued through the perito-
P R O C E D U R E S FOR R U M E N STUDIES
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Figure 4. First stage of Jarrett (1948) fistulation procedure: (A) exposed hernia in rumen wall being secured to skin by stay sutures, (B) tying the stay sutures over gauze roll, (C) stay sutures completed showing exposed hernia prior to closing incision, and (D) completed first stage.
neum and brought externally through the skin approximately 2.5 to 5.0 cm. from the incision line. The muscle layers and fascia are avoided so that the sutures retract them from the herniated area. When both ends of the suture silk have been brought through to the skin, the free exteriorized ends of the suture are firmly tied over a small wad of gauze previously steeped in a weak iodine solution (figure 4). After the stay sutures have been inserted, the pocket of the rumen or hernia will protrude slightly. The surface of the hernia is lightly scarified before the original incision is closed to promote healing and adhesion. To close the incision line, several stay sutures are inserted in such a way that the rumen is attached by the sutures. The remainder of the incision may be closed with a continuous line suture. Care should be taken during closure to insure that the two edges of the incision are everted and that
about 1 cm. of the original incision opening is left open to assure a drainage channel. The incision line and points of stay sutures are treated with antibiotic ointment and sprayed with plastic bandage. After 10 days the stay and incision line sutures may be removed. Symptoms of infection should be treated at that time and every 1 or 2 days thereafter until healing is complete. The second stage of the operation for insertion of the cannula can be performed as early as 10 to 12 days after the initial operation, but can be performed several months later if necessary. Prior to the insertion of the cannula in the second stage of the operation, the animal should again be fasted and the local area of the hernia cleaned and anesthetized with procaine. An incision is made in the hernia directly through the skin and the rumen wall. This incision should be approximately 2.5 cm. long for fitting the original Jarrett can-
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JOHNSON design described by Hentschl et al. (1954) is convenient for use with large openings such as are usually provided in bovines by the Schalk and Amadon (1928) procedure. Jarrett (1948) described a rubber cannula which is very economical and suitable for use with his surgical procedure. The original Jarrett cannula 5 has an inside diameter of approximately 26 mm. (figure 5). A cannula very similar in design, but somewhat larger in inside diameter, i.e., 38 ram., is also available 6 and may be more suitable for procedures requiring insertion of larger objects in the rumen. Plastic and rubber cannulas of almost any design, however, can be used successfully with the usual rumen fistula.
Other Fistulation Techniques
nula and should be started as close to the dorsal border of the hernia as possible. I t may be convenient, but not necessary, for insertion of the cannula to use retractors to hold the two surfaces of the incision apart and in an upward and outward direction. The cannula (figure 5) is wetted with a slightly soapy solution and literally folded inside out by directing as much of the flange as possible up the neck of the cannula, so that the remaining portion of the flange forms a small protrusion that may be inserted through the fistula opening. This portion of the flange is then inserted into the fistula as far as possible, and by means of gentle manipulation the rest of the cannula flange is unfolded back into its natural position inside the rumen. If the size of the incision is correct, the flange can usually be worked out completely into an unfolded or unkinked flat surface within the rumen. The area around the fistula opening should now be cleaned and treated with an antibiotic preparation. I t is most convenient to install the steel collar on the outside by inserting the nose of a pair of pliers through the opening in the steel collar to pick up the neck of the cannula. The steel collar may then be worked down around the pliers over the neck to the proper depth. The mouth of the cannula is then sealed with a stopper, and the operation is complete. Although the procedure was originally described for sheep, it has also been successfully used for cattle with openings for as large as a 10cm. cannula. I t is somewhat easier to get a tight fit using this procedure, since insertion of the cannula is made at the same time that the incision is made directly into the rumen. Cannula. Although several types of cannulas have been used in rumen fistulas, the
Esophageal fistulation techniques and canhulas have recently been improved to the extent that this technique has become highly useful in studying intake a n d digestibility of grazed forages. M a n y of the basic techniques and designs were developed b y Torell (1954), Bath et al. (1956), Cook et al. (1958), Lesperance et al. (1960), McManus (1961, 1962) and McManus et al. (1962). One of the better means to study activity in the lower portions of the digestive tract and their relation to the activities within the tureen is through the use of re-entrant cannulas. Although these procedures: present more of a challenge surgically and hence have not been used as much, they offer some of the few techniques available for this type of study. They have been used successfully by British workers (Phillipson, 1952 ; Hogan and Phillipson, 1960; Singleton, 1961; Ash, 1961a, b, 1962a, b) and by Dougherty (1955) in the United States. These workers not only described the techniques and some of their uses but also the molds for making the cannulas. The paper by Dougherty (1955) provides some excellent diagrams of fistulation operations for the rumen, duodenum, abomasalduodenal shunt and caecum, as well as descriptions of the construction of cannulas.
Rumen Volume Determination
Figure 5. Two sizes of Jarrett type cannulas shown with metal collars and rubber stoppers.
tralian Rubber Mills, Adelaide, South Australia. 6 Mould 5i1701, Avon India Rubber Company, Ltd., Melksham, Wiltshire, England.
In many studies of activity within the rumen, especially quantitative studies, it is de5The Jarrett cannula is obtainable from the South Aus-
PROCEDURES FOR R U M E N STUDIES sirable to determine the volume of the rumen. Perhaps the earliest technique and one which is still used occasionally is slaughter of the animal followed by ligation of the compartments and physical measurements of weight, volume, etc. The impracticality of slaughter techniques in most studies is obvious, however, and improved methods have been studied. Most of these depend on the use of nonabsorbable or nonmetabolizable materials. Swedish workers (Sperber et al., 1953; Hyden, 1955, 1960, 1961) employed polyethylene glycol as an inert marker in the determination of tureen fluid volume. This high molecular weight material is not absorbed from the rumen and can be determined analytically by gravimetric, turbidimetric and colorimetric techniques. Since its development, this technique has been widely used with varying degrees of success. Although the author has not used this method, users have suggested that the method does not yield reproducible data and is very time consuming. Recently, lithium salts have been used to determine liquid volume in the tureen. The lithium ion is virtually nonmetabolizable and is easily determined by flame photometric techniques on filtered rumen fluid. The procedure usually calls for administration of a dose of lithium sulphate orally and removal of a sample of rumen contents at some specified time after dosing for lithium determination. Although some workers (D. B. Purser, personal communication, Ohio State University) recommended sampling 1 hr. after dosing sheep, workers in the author's laboratory have determined that a more representative sample is obtained 2 ~ to 3 hr. after dosing beef cattle. Walker and Hawley (1965) compared lithium and polyethylene glycol in the determination of rumen volume and found cl0se agreement between the two methods on the first day after dosing. Lithium is absorbed, however, and partially recycled to the rumen via the saliva. The main disadvantage of the techniques described is that they are indicators primarily of the fluid volume and not necessarily of the solid ingesta volume. Attempts to develop indicators for the solid ingesta have not been as successful. Chromic oxide has been the indicator most widely used. Any solid material used as an indicator should theoretically have approximately the same specific gravity and particle size as the solid ingesta. Since these two qualities are usually highly variable with
871
most practical rations, the determination of this fraction is not easily made by indicator techniques presently available. Studies on the distribution of chromic oxide in the rumen have been reported by Corbett and Greenhalgh (1959) and Corbett et al. (1960a, b).
Gnotobiotics
The use of gnotobiotic animals in rumen studies is still in its infancy. Nevertheless, the determination of the function and importance of the rumen microorganisms in the ruminant will eventually depend on the use of this type of technique. The number of aspects of ruminant nutrition and physiology which can be studied with this type of animal presents a challenging array. T h e following represents only a partial listing: 1. The role and necessity of volatile fatty acids in the ruminant. 2. The utilization of natural proteins in a nonruminating animal and the relation to the nitrogen metabolism in the rumen microbial population. 3. The differential functions of any number of isolated bacterial species which might be added to the rumen in pure culture or in defined mixtures. 4. The differential function of protozoa and bacteria. 5. The requirements for the many micronutrients and growth factors known to be required by monogastric animals. Although the first "germ-free" ruminants were reported by Kuster (1912, 1913), little more was accomplished with this technique until the report by Luckey (1960). Considerable progress has been made recently toward the accomplishment of the techniques needed for such studies at Ohio State University and Michigan State University. 7 Such techniques begin with the delivery of newborn ruminants by Caesarean section under aseptic conditions and the maintenance of these young animals in isolation units in germ-free conditions. The mere maintenance of the animal has proved to be a challenge in itself, to say nothing of the imposition of experimental variables on an animal. Techniques for gnotobiotic research are most fully described by Luckey (1963).
7 The groups at Ohio State University and Michigan State University are under the leadership of D. B. Purser and C. Smith, respectively.
872 Summary
JOHNSON value of alfalfa and timothy by varied techniques. J. Dairy Sci. 44:2232. Ash, R. W. 1961a. Acid secretion by the abomasum and its relation to the flow of food material in sheep. J. Physiol. 156:93. Ash, R. W. 1961b. Stimuli influencing the secretion of acid by the abomasum of sheep. J. Physiol. 157: 185. Ash, R. W. 1962a. Omaso-abomasal re-entrant cannulae for sheep. J. Physiol. 164:4. Ash, R. W. 1962b. Gastro-intestinal re-entrant cannulae for studies of digestion in sheep. An. Prod. 4:309. Baker, F. and S. T. Harris. 1947. The role of the microflora of the alimentary tract of herbivora with special reference to ruminants. 2. Microbial digestion in the rumen (and caecum) with special reference to the decomposition of structural cellulose. Nutr. Abstr. Rev. 17:3. Baker, T. I., G. u Quicke, O. G. Bentley, R. Johnson and A. L. Moxon. 1959. The influence of certain physical properties of purified celluloses and forage celluloses on their digestibility by rumen microorganisms in vitro. J. Animal Sci. 18:655. Balch, C. C. 1950. Factors affecting the utilization of food by dairy cows. I. The rate of passage of food through the digestive tract. British J. Nutr. 4:361. Batch, C. C., and V. W. Johnson. 1950. Factors affecting the utilization of food by dairy cows. 2. Factors influencing the rate of breakdown of cellulose (cotton thread) in the rumen of the cow. British J. Nutr. 4:389. Barnes, R. F. 1966. Collaborative in vitro rumen fermentation studies on forage substrates. J. Animal Sci. (Submitted for publication). Barnett, A. J. G. and R. L. Reid. 1961. Reactions in the Rumen. Edward Arnold, London. Bath, D. L., W. C. Wier and D. T. Torell. 1956. The use of the esophageal fistula for the determination of consumption and digestibility of pasture forage for sheep. J. Animal Sci. 15:1166. Baumgardt, B. R., J. L. Cason and M. W. Taylor. 1962a. Evaluation of forages in the laboratory. I. Comparative accuracy of several methods. J. Dairy Sci. 45 : 59. Baumgardt, B. R., M. W. Taylor and J. L. Cason. 1962b. Evaluation of forages in the laboratory. II. Simplified artificial rumen procedure for obtaining repeatable estimates of forage nutritive value. J. Dairy Sci. 45 : 62. Baumgardt, B. R. and Hi Kon Oh. 1964. Evaluation of forages in the laboratory. IV. Within and among trial variability of the Wisconsin artificial rumen procedure. J. Dairy Sci. 47:263. Belasco, I. J., M. F. Gribbins and D. W. Kolterman. 1958. The response of rumen microorganisms to pasture grasses and prickly pear cactus following foliar application of urea. J. Animal Sci. 17:209. Bentley, O. G. 1959. A comparison of artificial rumen techniques. Report TID-7578, Oklahoma Conference, Radioisotopes in Agriculture, p. 181. Bentley, O. G., R. R. Johnson, S. Vanecko and C. H. Hunt. 1954a. Studies on factors needed by rumen microorganisms for cellulose digestion in vitro. J. Animal Sci. 13 : 581. Bentley, O. G., A. Lehmkuhl, R. R. Johnson, T. V. Hershberger and A. L. Moxon. 1954b. The "cellulolytic factor" activity of certain short-chained fatty acids. J. Am. Chem. Soc. 76:5000. Bentley, O. G., R. R. Johnson, T. V. Hershberger, J. H. Cline and A. L. Moxon. 1955. Cellulolytic
I n this r e v i e w the a u t h o r has a t t e m p t e d to discuss b o t h in vitro a n d in vivo t e c h n i q u e s for s t u d y i n g r u m e n m i c r o b i a l a c t i v i t y and function. F r o m the s t a n d p o i n t of the a d v a n tages a n d d i s a d v a n t a g e s of the v a r i o u s techniques, the p o t e n t i a l uses a n d in some cases the ease of m a n i p u l a t i o n , aspects of the in vitro techniques b a r e been described in detail to allow the r e a d e r to a s c e r t a i n w h i c h procedures m i g h t satisfy his r e q u i r e m e n t s . M o s t of the in vivo t e c h n i q u e s discussed were n o t described in sufficient d e t a i l to allow the r e a d e r to use them, b u t h o p e f u l l y to enable the r e a d e r to select the t e c h n i q u e s t h a t m i g h t seem m o s t useful a n d s u b s e q u e n t l y refer to the l i t e r a t u r e for f u r t h e r details. I n m o s t cases the descriptions offered b y the originators of the t e c h n i q u e s w o u l d be far m o r e a c c u r a t e a n d helpful t h a n those t h a t m i g h t be given b y the reviewer. I n selection of an in vitro t e c h n i q u e , there is no s u b s t i t u t e for logic i n v o l v i n g the obj e c t i v e of the s t u d y a n d the i n t e r p r e t a t i o n w h i c h is to be m a d e following the study. T h e s e factors m u s t be considered fully prior to t h e design of the e x p e r i m e n t . I n m a n y cases p u b l i s h e d t e c h n i q u e s will of n e c e s s i t y h a v e to be modified to satisfy the r e q u i r e m e n t s of the study. T o the m o r e sophisticated b i o c h e m i s t s some of the t e c h n i q u e s described m a y a p p e a r crude and l a b o r i o u s w h e n c o m p a r e d w i t h the m o r e refined b i o c h e m i c a l m e t h o d s a v a i l a b l e today. H o w e v e r , w i t h the c o m p l e x s y s t e m t h a t exists in r u m e n f e r m e n t a t i o n these t e c h n i q u e s m a y well serve as a p r e l i m i n a r y or a d j u n c t to m o r e refined studies. I n m a n y respects in vitro t e c h n i q u e s l e n d t h e m selves to w o r k w i t h radioisotopes, resting-cell a n d cell-free suspensions. I n addition, welld e v e l o p e d t e c h n i q u e s for s t u d y i n g b a c t e r i a in p u r e culture exist and can be p r o f i t a b l y utilized in c o n n e c t i o n w i t h s o m e of the studies outlined above. Literature Cited
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PROCEDURES
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factor activity of certain short-chain fatty acids for rumen microorganisms in vitro. J. Nutr. 57: 389. Blaxter, K. L. 1962. The Energy Metabolism of Ruminants. Charles C Thomas, Publ., Springfield, Illinois. Bowie, W. C. 1962. In vitro studies of rumen microorganisms using a continuous-flow system. Am. J. Vet. Res. 23:858. Bryant, M. P. 1959. Bacterial species of the rumen. Bact. Rev. 23:125. Bryant, M. P. and R. N. Doetsch. 1955. Factors necessary for the growth of Bacteroides succinogenes in the volatile acid fraction of tureen fluid. J. Dairy Sci. 38:340. Burroughs, W., N. A. Franks, P. Gerlaugh and R. M. Bethke. 1950a. Preliminary observations upon factors influencing cellulose digestion by tureen microorganisms. J. Nutr. 40:9. Burroughs, W., H. G. Headley, R. M. Bethke and P. Gerlaugh. 1950b. Cellulose digestion in good and poor quality roughages using an artificial rumen. J. Animal Sci. 9:513. Burton, G. W., F. E. Knox and D. W. Beardsley. 1964. Effect of age on the chemical composition, palatability and digestibility of grass leaves. Agron. J. 56:160. Campling, R. C., M. Freer and C. C. Balch. 1961. Factors affecting the voluntary intake of food by cows. 2. The relationship between the voluntary intake of roughages, the amount of digesta in the reticulo-rumen and the rate of disappearance. British J. Nutr. 15:531. Castle, Elizabeth J. 1956. The rate of passage of foodstuffs through the alimentary tract of the goat. I. Studies on adult animals fed on hay and concentrates. British J. Nutr. 10:15. Chandler, P. T., E. M. Kesler and R. D. McCarthy. 1964. Polyethylene as a reference substance for digestion studies with young ruminants. J. Dairy Sci. 47:1426. Cheng, E. W., G. Hall and W. Burroughs. 1955. A method for the study of cellulose digestion by washed suspensions of rumen microorganisms. J. Dairy Sci. 38:1225. Clarke, R. T. J. 1963. The cultivation of some rumen oligotrich protozoa. J. Gen. Microbiol. 33:401. Coleman, G. S. 1960. The cultivation of sheep rumen oligotrich protozoa in vitro. J. Gen. Microbiol. 22: 555. Coleman, G. S. 1962. The preparation and survival of bacteria-free suspensions of Entodinium caudatum. J. Gen. Microbiol. 28:271. Cook, C. W., J. L. Thorne, J. T. Blake and J. Edlefsen. 1958. Use of an esophageal-fistula cannula for collecting forage samples by grazing sheep. J. Animal Sci. 17 : 189. Corbett, J. L. and J. F. D. Greenhalgh. 1959. Distribution of Cr~O~ and P. E. G. in the rumenretieulum of cattle. British J. Nutr. 13:337. Corbett, J. L., J. F. D. Greenhalgh, I. McDonald and E. Florence. 1960a. Excretion of chromium sesquioxide administered as a component of paper to sheep. British J. Nutr. 14:289. Corbett, J. L., G. W. Reid, J. P. Langlands and E. Florence. 1960b. Further studies on the administration of chromium sesquioxide as a component of paper. Proc. Nutr. Soc. 19:xx. Cuthbertson, D. P. 1963. Progress in Nutrition and Allied Sciences. Oliver and Boyd, Edinburgh. Davey, L. A., G. C. Cheeseman and C. A. E. Briggs.
1960. Evaluation of an improved artificial rumen designed for continuous control during prolonged operation. J. Agr. Sci. 55:155. Dehority, B. A. 1961. Effect of particle size on the digestion rate of purified cellulose by rumen cellulolytic bacteria in vitro. J. Dairy Sci. 44:687. Dehority, B. A., K. el-Shazly and R. R. Johnson. 1960. Studies with the cellulolytic fraction of rumen bacteria obtained by differential centrifugation. J. Animal Sci. 19:1098. Dehority, B. A. and R. R. Johnson. 1961. Effect of particle size upon the in vitro cellulose digestibility of forages by rumen bacteria. J. Dairy Sci. 44: 2242. Donefer, E., E. W. Crampton and L. E. Lloyd. 1960. Prediction of the nutritive value index of a forage from in vitro rumen fermentation data. J. Animal Sci. 19:545. Dougherty, R. W. 1955. Permanent stomach and intestinal fistulas in ruminants: some modifications and simplifications. Cornell Vet. 45:331. Dougherty, R. W., R. S. Allen, W. Burroughs, N. L. Jacobson and A. D. McGilliard. 1965. Physiology of digestion in the ruminant. Proc. Second Intern. Symposium on Physiology of Digestion in the Ruminant. Butterworth, Inc., Washington, D. C. Eadie, J. M. 1962a. The development of tureen microbial populations in lambs and calves under various conditions of management. J. Gen. Microbiol. 29:563. Eadie, J. M. 1962b. Inter-relationships between certain rumen ciliate protozoa. J. Gen. Microbiol. 29: 579. el-Shazly, K., B. A. Dehority and R. R. Johnson. 1960. A comparison of all-glass, semipermeable membrane and continuous flow type of apparatus for in vitro rumen fermentations. J. Dairy Sci. 43: 1445. el-Shazly, K., B. A. Dehority and R. R. Johnson. 1961a. Effect of starch on the digestion of cellulose in vitro and in vivo by tureen microorganisms. J. Animal Sci. 20:268. el-Shazly, K., R. R. Johnson, B. A. Dehority and A. L. Moxon. 1961b. Biochemical and microscopic comparison of in vlvo and in vitro rumen fermentations. J. Animal Sci. 20:839. Erwin, E. A. and N. G. Elliston. 1959. Rapid method of determining digestibility of concentrates and roughages in cattle. J. Animal Sci. 18:1518. (Abstr.). Fina, L. R., G. W. Teresa and E. E. Bartley. 1958. An artificial tureen technique for studying tureen digestion in vivo. J. Animal Sci. 17:667. Fina, L. R., C. L. Keith, E. E. Bartley, P. A. Hartman and N. L. Jacobson. 1962. Modified in vivo artificial rumen (VIVAR) techniques. J. Animal Sci. 21:930. Gray, F. V., R. A. Weller, A. F. Pilgrim and G. B. Jones. 1962. A stringent test for the artificial rumen. Australian J. Agr. Res. 13:343. Gutierrez, J. 1955. Experiments on the culture and physiology of holotrichs from the bovine rumen. Biochem. J. 60:516. Gutierrez, J. and R. E. Davis. 1959. Bacterial digestion by the rumen ciliates E n t o d l n i u m and Diplodinium. J. Protozool. 6:222. Gutierrez, J., and R. E. Davis. 1962. Culture and metabolism of the rumen ciliate Epidinium encand a t u m (Crawley). Appl. Microbiol. 10:305. Hall, G., E. W. Cheng and W. Burroughs. 1953. Bvitamins and other factors stimulatory to cellulose
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JOHNSON Johnson, R. R., B. A. Dehority and O. G. Bentley. 1958. Studies on the in vitro rumen procedure: improved inoculum preparation and the effects of volatile fatty acids on cellulose digestion. J. Animal Sci. 17:841. Johnson, R. R., B. A. Dehority, H. R. Conrad and R. R. Davis. 1962a. Relationship of in vitro cellulose digestibility of undried and dried mixed forages to their in vivo dry matter digestibility. J. Dairy Sci. 45:250. Johnson, R. R., B. A. Dehority, J. L. Parsons and H. W. Scott. 1962b. Discrepancies between grasses and alfalfa when estimating nutritive value of forages from in vitro cellulose digestibility by rumen microorganisms. J. Animal Sci. 21:892. Johnson, R. R., B. A. Dehority, K. E. McClure and J. L. Parsons. 1964. A comparison of in vitro fermentation and chemical solubility methods in estimating forage nutritive value. J. Animal Sci. 23: 1124. Kamstra, L. D., A. L. Moxon and O. G. Bentley. 1958. The effect of stage of maturity and lignification on the digestion of cellulose in forage plants by rumen microorganisms in vitro. J. Animal Sci. 17:199. King, K. W. and W. E. C. Moore. 1957. Density and size as factors affecting passage rate of ingesta in the bovine and human digestive tracts. J. Dairy Sei. 40:528. Kuster, E. 1912. Die Keimfreie Ziichtun~ yon S~iugefieren und ihre Bedeutung fur die Erforschung der K6rperfunktionen. Sentr. Bakterial. Parasitenk. 54:44. Kuster, E. 1913. Die Gewin'nung und Ziichtung Keimfreier Sangetiere. Dent. Med. Wochschr. 39:1586. Lesperance, A. L., V. R. Bohman and D. W. Marble. 1960. Development of techniques for evaluating grazed forage. J. Dairy Sci. 43:682. Lewis, D. 1961. Digestive Physiology and Nutrition of the Ruminant. Butterworths, London. Luckey, T. O. 1960. Germ-free lamb nutrition. Abstr. Fifth Intern. Congr. Nutr., Washington, D. C. p. 25. Luckey, T. O. 1963. Germ-free Life and Gnotobiology. Academic Press, New York, N. Y. Lusk, J. W., C. B. Browning and J. T. Miles. 1962. Small-sample in vivo cellulose digestion procedure for forage evaluation. J. Dairy Sci. 45:69. Mah, R. A. 1964. Factors influencing the in vitro culture of the rumen ciliate Ophryascolex purkynei Stein. J. Protozool. 11 : 546. Mah, R. A. and R. E. Hungate. 1965. Physiological Studies on the rumen ciliate, Ophryascolex purkynei Stein. J. Protozool. 12:131. Marston, H. R. 1948. The fermentation of cellulose in vitro by organisms from the rumen of sheep. Biochem..l. 42:564. McAnally, R. A. 1942. Digestion of straw by the ruminant. Biochem. J. 36:392. McAnally, R. A. 1943. Studies on the alimentary tract of Merino sheep in South Africa. X. Notes on digestion of some sugars in the rumen of sheep. Onderstepoort J. Vet. Sci. An. Ind. 18:131. McDougall, E. I. 1949. Studies on ruminant saliva. I. The composition and output of sheep's saliva. Biochem. 5.43: 99. McManus, W. R. 1961. Properties of roughage feedstuffs collected from oesophageal fistulas. Australian J. Exp. A~r. An. Husb. 1:159. McManus, W. R. 1962. Oesophageal fistulation studies in the sheep. Australian Vet. J. 38:85.
digestion by washed suspensions of rumen microorganisms. J. Animal Sci. 12:918. Hall, O. G., H. D. Baxter and C. S. Hobbs. 1961. Effect of phosphorus in different chemical forms on in vitro cellulose digestion by rumen microorganisms. J. Animal Sci. 20:817. Harbers, L. H. and A. D. Tillman. 1962. Continuous liquid culture of rumen microorganisms. J. Animal Sci. 21:575. Hentschl, A. F., R. N. Berry and C. F. Huffman. 1954. A plastic plug for use in the bovine rumen fistula. Mich. State Coll. Vet. 14:77. Hershberger, T. V. and E. W. Hartsook. 1960. Studies on the fermentation of alfalfa hay by rumen microorganisms in vitro. Carbon dioxide, methane and heat production. Federation Proc. 19:321. (Abstr.). Hershberger, T. V., T. A. Long, E. W. Hartsook and R. W. Swift. 1959. Use of the artificial rumen technique to estimate the nutritive value of forages. J. Animal Sei. 18:770. Hobson, P. N., and W. Smith. 1963. Continuous culture of rumen bacteria. Nature. 200:607. Hoflund, S., J. I. Quin and R. Clark. 1948. Studies on the alimentary tract of Merino sheep in South Africa. XV. The influence of different factors on the rate of cellulose digestion (a) in the rumen and (b) in ruminal ingesta as studied in vitro. Onderstepoort J. Vet. Sci. An. Ind. 23:395. Hogan, J. P. and A. T. Phillipson. 1960. The rate of flow of digesta and their removal along the digestive tract of sheep. British J. Nutr. 14:147. Hopson, J. D., R. R. Johnson and B. A. Dehority. 1963. Evaluation of the dacron bag technique as a method for measuring cellulose digestibility and rate of forage digestion. J. Animal Sci. 22:448. Hubbert, F., Jr., E. Cheng and W. Burroughs. 1958a. Mineral requirement of rumen microorganisms for cellulose digestion in vitro. J. Animal Sci. 17:559. Hubbert, F., Jr., E. Cheng and W. Burroughs. 1958b. The influence of potassium, sodium, rubidium, lithium and cesium on in vitro cellulose digestion by rumen microorganisms with observations upon sodium and potassium influences in lamb fattening rations..l. Animal ScL 17:576. Hungate, R. E. 1950. The anaerobic mesophilic cellulolytie bacteria. Bact. Rev. 14:1. Hungate, R. E. 1955. Mutualistic intestinal protozoa. In Biochemistry and Physiology of Protozoa, Vol. II (Edited by S. H. Hutner and A. Lwoff). Acad. Press, New York. Hungate, R. E., M. P. Bryant and R. A. Mah. 1964. The rumen bacteria and protozoa. Ann. Rev. Microbiol. 18:131. Hyden, S. 1955. A turbidimetric method for the determination of higher polyethylene glycols in biological materials. Ann. Roy. Agr. Coll. Sweden. 22:139. Hyden, S. 1960. The use of reference substances and the measurement of flow in the alimentary tract. Digestive Physiology and Nutrition of the Ruminant. Proc. Univ. of Nottingham Seventh Easter School in Agr. Sci. p. 35. Hyden, S. 1961. Determination of the amount of fluid in the reticulo-rumen of the sheep and its rate of passage to the omasum. Ann. Roy. Agr. Coll. Sweden. 27:51. Jarrett, I. G. 1948. The production of rumen and abomasal fistulae in sheep. J. Council Sci. Ind. Res. Australia. 21:311. Johnson, R. R. 1963. Symposium on microbial digestion in ruminants: In vitro rumen fermentation techniques. J. Animal Sci. 22:792.
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Techniques and Procedures for In Vitro and In Vivo Rumen Studies Ronald R. Johnson J ANIM SCI 1966, 25:855-875.

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TECHNIQUES AND PROCEDURES FOR IN VITRO AND I N VIVO RUMEN S T U D I E S 1, 2

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JOHNSON Cuthbertson (1963), Lewis Dougherty et al. (1965). (1961) and

Reviews covering early developments with

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BUNSEN VALVE IN VITRO

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When the large populations of protozoa

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