J Anim Sci 1966 Johnson 855 75

 

Techniques and Procedures for In Vitro and In Vivo Rumen Studies Ronald R. Johnson

 J ANIM SCI SCI

1966, 25:855-875.

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TECHNIQUES AND

AND

IN

PROCEDURES FOR VIVO R U M E N STU DIE S RONALD RONA LD R.

IN

VITRO

1, 2

JOHNSON 3

Page

INTRODUCTION V I T R O TECHNIQUES History Potential Uses of I n V i t r o Techniques Contin uous Flow Systems Closed System s Description of Techniques Buffer Solutions and Nutri tional Media Ferm entat ion Ves Vessel selss Degree of Agitat ion and Gas Phase Temperature Optim um pH Inoc ulum Source Sourcess F e rm rm en e n t at at i on on T im im e P er er io io ds ds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Termi natin g the Ferm entat ion Analytic al Proce dures Interpretation of I n V i t r o Results Rume n Protozoo logy P ur u r e C ul ul tu tu re re T ec ec hn hn iq iq ue ue s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IN V I V O TECHNIQUES VIV AR Techniques Nylo n Bag Techniques Rate of Passage Measure ments Rum en Fistulat ion Techniques Schalk and Amado n Procedure J a rr r r e tt tt P ro r o ce ce du du re re . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Cannula O th th er er F is is tu tu la la ti ti on on T ec ec hn hn iq iq ue ue s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . R u me m e n V ol ol um um e D et e t er e r mi m i n at at i on on . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G no no to to bi bi ot ot ic ic s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . , .... .... .... .... .... .... .... .... .... ... SUMMARY LIT ERAT URE CITED IN

Introduction HE ruminant animal has often been classified 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, c a n v a r y i n d e p e n d e n t l y a n d c e r t a i n ly l y p o ss ss e ss ss different metabolic characteristics. Inherently the rumin ant anim al generally has the same type of nutrient requirements as most other s p e c i e s o f l a r g e a n i ma l s , i . e . , b a s i c r e q u i r e ments for energy, protein with certain bal1 Approved for publication as Journal Article No. 82-6 5 by the Associate Directo Directo~ ~ of the Ohi o Agricultural Research and Development Center, Wooster, Ohio. Paper preoared for a revision of the monograph, Technlaues and Procedures in Ani Animal mal Production Research. Research. z Department of Animal Science, Ohio Agricultural Agricultural Research and Developme Development nt Center, Center, Wooster, Ohio. Ohio.

:

855 856 856 856 857 85 7 857 85 7 857 85 7 857 85 7 858 85 8 859 85 9 860 860 86 0 86l 862 863 86 3 863 86 3 863 864 86 4 865 865 865 865 86 5 866 86 6 867 86 7 867 86 7 868 870 870 870 871 872 872

a n c e s o f a m i n o a c i d s, s, m i n e r a l s , v i t a m i n s i n a l l t h e c l a s s e s , e s s e n t i a l f a t t y a c i d s , e t c . Me t a bolically speaking, the greate st 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 anim al s perfo rma nce both in nat u r e a n d e f f ic ic i e n c y a n d i n a d d i t i o n i s u n d o u b t edly directly related to the endocrinological control of energ y metabolism. W ith the except i o n o f t h i s m a j o r d i f fe fe r e n c e i n t h e a n i m a l s t i s s u e s per se t h e o t h e r m a j o r c o m p l i c a t i n g factor in studying ruminant nu trition and m etabolism 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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within their digestive tracts, only ruminants, species with an enlarged or more functional caecum, and certain marsupials can claim a m ajor reliance on the microorganisms for their nutritional welfare. Although the metabolic system within the ruminant animal itself and the metabolic system in the m icrobial popu lation definitely affect each other in many ways, they are also capable of varying almost independently in s o me c h a r a c t e r i s t i c s . As a c o n s e q u e n c e r e f i n e d studies of rumen microbial activity in the intact ruminant animal are subject to tremend o u s v a r ia i a t i o n s . R e c o g n i t i o n o f t h is is f a c t m a n y y e a r s a g o p r o m p t e d i n i t i a ti ti o n o f s t u d i e s o f t h e activity of rumen microorganisms by several techniques. Although those working most c l o s e ly ly w i t h t h e s e t e c h n i q u e s w i ll ll r e a d i l y a d mit they leave something to be desired, it can be truthfully said these techniques have evolved to a point where they are worthy of r e v i e w i n a s in in g l e p a p e r f o r t h e u s e o f i n v e s t i gators 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 d i v i d e d i n t o t w o m a i n c a t e g o r i e s: s : ( 1 ) in vitro t e c h n i q u e s a n d ( 2 ) in vivo t e c h n i q u e s . T h e in vitro t e c h n i q u e s d e p e n d u p o n c o m p l e t e r e moval of the microorganisms from the control o r i n f l u e n c e o f t h e h o s t a n i ma l i t s e l f , wh i l e t h e in vivo t e c h n i q u e s i n v o l v e e i t h e r a p a r t i a l c o n trol of environmental effects or possibly q u a n t i t a t i o n o f t h e e f f e c ts ts w i t h i n t h e a n i m a l . 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 f a c t o r s wh i c h a f f e c t t h e m . I n t h i s d i s c u s s io io n in vitro t e c h n i q u e s w i l l b e c o n s i d e r e d i n d e t a i l f i r s t , a n d s o me d i s c u s s i o n o f v a r i o u s in vivo t e c h n i q u e s wi l l f o l l o w. I t wi l l b e i mp o s s i b l e t o describe in detail all of the useful techniques that have been developed in various laborat o r i e s . B y n e c e s s i t y t h e d i s c u s s i o n wi l l b e i n f l u e n ce ce d g r e a t l y b y t h e a u t h o r s p e r s o n a l e x perience, 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 a c t i v i t y t h e s t u d e n t i s r e f e r r e d t o e a r l y r e v i ew ew s by Baker and Harris (1947), McNaught and S m i t h ( 1 9 4 7 ) , P h i ll l l ip ip s o n ( 1 9 4 7 ) a n d M a r s t o n (1948) and to more recent comprehensive treatments of the subject by Annison and Lewis (1959), Barnett and Reid (1961),

Cuthbertson (1963), Lewis D o u g h e r t y et al ( 1 9 6 5 ) .

(1961)

and

In Vitro Techniques History

Reviews covering early developments with in vitro i n v e s t i g a t i o n s h a v e b e e n w r i t t e n b y Mo xon and Bentley (1955) and Ben tley ( 1 9 5 9 ) . M o r e r e c e n tl tl y J o h n s o n ( 1 9 6 3 ) h a s presented a comprehensive discussion of the h i s t o r i c a l d e v e l o p m e n t o f in vitro r u m e n f e r mentation techniques and their uses for studying rumen microbial activity. In the developm e n t a n d s e l e c t i o n o f a n in vitro t e c h n i q u e for studying rumen fermentation, the investigator must decide initially whether his objective is to duplicate as closely as possible the actual ferm entation occurring within the rumen of the animal or merely to study qualitat i v e l y a n d q u a n t i t a t i v e l y a f e w o f th th e m a n y p r o c e s s e s o c c u r r i n g a s a r e s u l t o f mi c r o b i a l activity. Potential Uses o f I n V i t r o Techniques

In an earlier review Johnson (1963) conc l u d e d t h a t in vitro t e c h n i q u e s c o u l d b e u t i l ized in the investigation of several problems: 1. Cellulose digestion an d factor s affecting it. 2. Utilization o f no nprote in nitrogen. 3 . I n t e r m e d i a t e m e t a b o l i sm sm i n b o t h m i x e d and pure cultures. 4. Studies of sym biosis utilizing bo th all glass systems and continuous flow chemostats. 5 . S t u d i es es o f r a t e p h e n o m e n a r e q u i r i n g a nonsteady state situation. 6. Forage evaluation studies. 7 . S t u d i e s o f b i o e n e r g e t i c s o f t h e r u me n f e r mentation as have been investigated by M arston (1948), Hershberger and Hartsook (1960) and Walker and Forrest (1964). Undoubtedly many other uses could be a d d e d t o t h i s l i s t ; h o we v e r , t h i s r a n g e i s s u f f i c i e n t i n i t s e l f t o c h a l l e n g e mo s t i n v e s t i g a t o r s o f r u m e n m i c r o b ia i a l a c t iv iv i t y . T h e m a i n a d v a n t a g e o f t h e in vitro t e c h n i q u e s r e s t s i n t h e ability to use them to study activity of microo r g a n i s m s a w a y f r o m t h e c o n t r o l a n d i n f l u en en c e imposed by the host animal. Further discussion of the advantages and disadvantages will be made in the comparison of the complex

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continuous flow techniques with the closed system techniques.

is its complex ity and the inability to perform large numbers of experiments with it.

Continuous Flow Systems

Closed Systems

If the attempt is to simulate actual processes within the rumen, the criteria which s h o u l d b e s a ti ti s fi fi e d h a v e b e e n l i s te te d b y W a r n e r ( 1 9 5 6 ) a n d D a v e y et al. ( 1 9 6 0 ) . T h e t e c h niques and apparatus designed to satisfy these requirements are considerably more complex than those required for the other type of obj e c t iv iv e . S u c h s y s t e m s k n o w n a s c o n t i n u o u s f lo lo w a p p a r a t u s o r c h e m o s t a t s h a v e b e e n d e s ig ig n e d a n d s t u d ie ie d b y W a r n e r ( 1 9 5 6 ) , D a v e y et al. ( 1 9 6 0 ) , G r a y et al. ( 1 9 6 2 ) , B o w i e ( 1 9 6 2 ) , A d l e r et al. ( 1 9 5 8 ) , H a r b e r s a n d T i ll ll m a n ( 1 9 6 2 ) , Q u i n n ( 1 9 6 2 ) , R u f e n e r et al. ( 1 9 6 3 ) , S t e w a r t et al. ( 1 9 6 1 ) , S l y t e r et al. ( 1 9 6 4 ) a n d H o b s o n a n d S m i t h ( 1 9 6 3 ) . I n t h e s e t e c h n i q u e s p r o v i s i o n in in t h e a p p a r a tus invariably has to be made for regular add i t io io n s o f n u t r i e n t s i n s o m e w h a t t h e s a m e w a y 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, i f s y s te te m s c a p a b l e o f s a t i s f y in in g t h e t h e o r e t i c a l requirements could be adopted, would enable quantitative calculations of certain microbial processes. When properly designed, conducted and int e r p r e t e d , t h e c o n t i n u o u s f lo lo w o r c h e m o s t a t 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 microb i a l c u l t u r e . T o a c c o m p l i s h t h is i s v e r y e x a c ti ti n g control must be maintained on nutrient input, e n d - p r o d u c t r e m o v a l , p H , n u t r i e n t c o n c en en tration, oxidation-reduc tion potential, rate of agitation, etc. Even with this type of control seemingly accomplished, the technique would b e o p e n t o s e v e r e c r i ti ti c i s m w i t h o u t c a r e f u l a s sessment 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 as tl illyl i on ptehnes a trheea ot ef cshynni tqhuees i tso- a cb rsiot ir cpitsio i om,n set supdei ce is. s-. T h e o t h e r c h i e f d i s a d v a n t a g e o f t h is is t e c h n i q u e

In contrast, the systems designed for meeting the second objective, i.e., quantitation of au l faet iwo np ,r oacrees smes e sa rokcecdu rbryi n sgi m i nptlhi ce i tmy i corfo bt hi ae li rp od pe -sign 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 rum en population in the intact animal. This undoubtedly should be the concern of anyone making full use of i n v i t r o t e c h n i q u e s . A s r e v ie ie w e d b y J o h n s o n ( 1 9 6 3 ) , however, a series of papers from Ohio (Deh o r i t y e t al. 1 9 6 0 ; e l - S h a z l y et al. 1 9 6 1 a , b ) have demonstrated quite well that bacteria propagated in vitro can be truly representatt hi vee a os sf utmh op st ieo ni n c tahne bien tma ca td er utm h aetn t hi tes ealcf t. i vTiht ui ess, 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 met a b o l i c p r o c e s s e s , s i n c e t h i s i n f a c t ma y e n hance 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 e l i mi n a t i o n o f p r o t o z o a f r o m t h e p o p ~ J l a t i o n . The role of protozoa in rumen microbial fermentations will simply have to be studied by other techniques. Description of Techniques B u f f e r S o l u t io io n s a n d N u t r i t i o n a l M e d i a . Although many researchers have arrived at a s t a n d a r d b u f f e r e d m e d i u m f o r t h e i r in v itr o f e r m e n t a t i o n t e c h n iq iq u e s t h r o u g h e x p e r i m e n t a l studies, most of the media are based on soc a l le le d M c D o u g a t l ' s s o l u t io i o n , w h i c h is is a n a r t if i f i c ia i a l s a li li v a o r b u f f e r m e d i u m b a s e d o n t h e

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a n a l y s is is o f s h e e p s a l i v a b y M c D o u g a l l ( 1 9 4 9 ) . M a n y o f t h e p r o c e d u r e s s p e c if i f y t h a t t h i s s o l uution alone is the buffer medium used in their in vitro fermentation studies. Others have added modifications for particular reasons. B u r r o u g h s et al. ( 1 9 5 0 a , b ) i n o n e o f t h e earlier in vitr o fe rme n ta tio n stu d ie s u se d M c D o u g a l l s s o l u ti t i o n m o d i f ie ie d b y t h e a d d i t i o n o f a s e r ie ie s o f t r a c e m i n e r a l s . S o m e w o r k e r s s u c h a s D o n e f e r e t a l. l. 1 9 6 0 ) h a v e i n c r e a s e d the buffering capacity of the solution, in order t o e l im i m i n a t e t h e n e c e s s i t y of of a d j u s t i n g p H i n the middle of a ferm entation run. O thers have varied the proportions of phosphates and bicarbonates as part of the buffering system. A n d e r s o n et al. ( 1 9 5 6 ) a n d H a l l et al. ( 1 9 6 1 ) used an in vitro rumen fermentation system for studying the availability and utilization o f p h o s p h o r u s i n v a r i o u s p h o s p h o r u s s u p p l eements 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. ( 1 9 5 4 a , b , 1 9 5 5 ) a n d B r y a n t a n d D o e t s c h (1955), valeric acid and biotin have become standard ingredients of the in vitro fermen tat io i o n m e d i a u s e d i n o u r l a b o r a t o r y w h e n c e ll ll u lose 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. T h e c o m p o s i t i o n o f M c D o u g a l l s s o l u t i o n is 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 v a r ie ie d i n a n y n u m b e r o f w a y s . 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 SAL IVA a

ARTIFICIAL

G m . / l i te te r o f d i s ti t i l le le d H ~ O

Ingre dient

NaHCO3 KC1 CaCI~ Na2HPO4 12 H~O

9.80 0.57 0.04 9. 30

NaC1 MgSO4 7 H_~O

0.47 0. 12

a McD ougall (1949).

TABLE

2. COM POSITION OF OHIO FERMENTATION MEDIA Ingredient

N

V TRO

M1./100 ml.

Na2CO3, 200 mg./m l. Mineral mixture ~ F e C h , 4 .4 .4 m g . / m l . C a C h , 5 .2 .2 9 m g . / m l .

1.0 2.0 1.0 1.0

U r e a , 1 26 m g . / m l . Biotin,b I0 mcg ./ml. V al eri c aci d ,b 5 m g ./ m l .

1.0 2.0 5.0

a T h e m i n e r a l m i x t u r e c o n s i s ts ts o f t h e f o l lo lo w i n g d is is s o l ve ve d and diluted to 1 liter in water: Na~HPO4, 56.5 gin.: NaH~PO4, 54.5 gin.: KCI, 21.5 gm.; NaC l, 21.5 gm.; M gSO~'7 H20, 5.82 g m . ; a n d K 2 S O~ O~ , 7 . 5 0 g i n . b B i o t i n a n d v a l e r i c a c i d h a v e b e e n s h o w n t o b e e s s e n t ia ia l only for cellulolytie rumen bacteria, but are added routinely in this laboratory in most media.

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 c a n b e a c r i t i c a l f a c to to r , e s p e c i a l l y w h e n b o t h starch- and cellulose-type substances are being digested in the same fermentation (elS h a z l y et al. , 1 9 6 1 a ) . W h e n w a s h e d c e l l i n ocula 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, mo st of the trace growth factor requirements are supplied therein. Fermentation Vessels. The apparatus for t h e c o n t i n u o u s f lo lo w o r c h e m o s t a t t y p e i n v i t r o rumen fermentations will not be described in this paper, and the reader is referred to the references cited. For the closed-system typ e fermentation, practically any type of vessel m a y b e u s e d ( e l - S h az a z l y et al. , 1 9 6 0 ) . T w o t y p i c a l s y s t e m s a r e s h o w n i n f ig ig u r e 1 . G e n erally speaking all-glass systems have been used by most workers in recent years. In our laboratory fermentation vessels varying from 1 0 t o 1 ,0 0 0 m l . i n f l u i d v o l u m e h a v e b e e n u t i l ized, 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 syste m which wilt involve the least possible number of transfers of fiber-containing media prior to final analysis. For example, the s u b s t r a t e i n q u e s t io i o n m a y b e w e i g h ed ed d i r e c t l y into a centrifuge tube which is also used as tfhe er m feenrtm a teinotna tti iomn e vt he ses et ul .b eAct a nt hbee ecne dn t roi ff u gt eh de and the supernatant discarded. Further analy-

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proper volume. For example, D ehority 1961) and Deho rity and Johnson 1961) removed samples at different times from the same 175m l . f e r m e n t a t i o n b o t t l e b y c a l i b r a t in in g t h e b o t tle at 100, 125, 145, 160 and 175 ml. Thus, s a m p le s o f 1 5 , 1 5 , 2 0 , 2 5 a n d 2 5 m l. c o u ld b e t a k e n a t s u c ce c e s s iv iv e t im im e s d u r i n g f e r m e n t a t i o n .

ianvgo ti od pr eem r mo ivte dp aws si tahg ea po ifp ef it bt er owu si t hp aar tl iacrlgees .o pTeon in excessive contact with oxygen during this sample removal it is convenient to prepare a rubber stopp er with a gassing tube extending to t h e b o t t o m o f a f e r m e n t a t i o n f l a s k o r b o t t le le . 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

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. E i t h e r m e t h o d d e s c r ib ib e d i s e q u a l l y a p p r o priate for studying other substrates as well. For example, the study of digestion of other c a r b o h y d r a t e s u b s t r a te te s m a y b e s o m e w h a t 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. I n v i t ro t e c h n i q u e s h a v e a l s o b e e n u s e d t o s t u d y 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 r e l a t i v e l y i n s o lu lu b l e . I n s u c h a c a s e t h e y u s u ally settle to the bottom of the fermentation v e ss ss e l a n d a r e n o t r e a d i l y s a m p l e d b y a p i p e t ring technique. For this reason the sample t a k e n a t z e r o t i m e a n d a t a l a t e r t im im e m a y n o t be representative of the same continuous ferm e n t a t i o n . T h i s c a n b e r e a d i l y s o lv lv e d b y u s in in g i n d i v id id u a l f e r m e n t a t i o n s f o r e a c h t i m e s a m p l e . D e g r e e o / A g i t a t i o n a n d G a s P h a s e I n v i tr tr o rumen fermentations have been conducted both with and without mechanical agitation. It is the opinion in many laboratories that agitation, ev en with insoluble substrate s, is n o t g e n e r a l l y n e c e s s a ry r y . U n p u b l i s h e d r e s u l ts ts f r o m o u r l a b o r a t o r y i n d ic i c a t e t h a t v i o l e n t ag ag i tation is actually deleterious to the digestion of cellulose. It has been common practice in most techniques to insure a complete mixing a t t h e t i m e o f in i n o c u l a ti ti o n a n d a t t h e t i m e m a terial is added to or removed from the flask. If the flask is continu ously g assed with carbon dioxide, this also provides a mild movement within the liquid medium of the fer-

oo cn ctuhres bboetttw l eese ins snaem c epsl si an rgys .t oH feancci el i, t actael idbirl au tt ii oo nn of the fermentation medium back to the

m s hoi fc hg ar es sc ionggs yesnt teamt iso nh a vv ee s sbeele. n Tuws oe d m , ba iont h t yopf ew nize the strict requirement for anaerobiosis for

UNSEN

VALVE

IN

V I TRO

FERMENTATION Figu Fi gure re I.

A) In

TUBE vitro

fermentation

tube

sBu h onws einn g v sa to tlv o pep es hr ofwo in r g a sosuin ith fCO CoO tleg t ws lit r ~e sacnadp e B) 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 m echanical stirrer or by hand, while a sample is being

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the proper culture of rumen bacteria. The first technique is a continuous gassing in which CO2 is bub bled slow ly throu gh the medium by placing a gassing tube near the bottom of the fermentation vessel. The second technique provides for an initial thorough satu r a t i o n o f t h e m e d i u m w i t h C O z f o ll ll o w e d b y

to them for significant periods of time, a loss in activity is often noticed. O p t i m u m p t t Since the buffering capacity of the medium itself is the only means by which pH can be controlled internally in an a l l -g - g l a s s s y s t e m , th t h e p H i n th th e s e s y s t e m s o f t e n changes during the process of fermentation.

c l o si s i n g th th e f e r m e n t a t i o n v e s s e l w i t h a s t o p p e r c o n t a i n i n g a B u n s e n v a l v e f i gu g u r e 1 ). ). T h e l a t t e r i t e m i s u su su a l l y n o m o r e t h a n a n i n v e r t e d rubber policeman placed over an outlet tube. A small slit cut in the rubber of the policeman a l l o w s g a s t o e s ca ca p e , b u t a i r i s p r e v e n t e d f r o m 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 probab ly more advisable if several serial samples are to be taken at intervals during fermentation, since it insures the maintenance of a CO2 atmos phe re at all times. However, this technique has the disadvantage

It is quite likely that this will have to be adjusted. The pH optimum for cellulose digestion has generally been found to be around 6 . 9 . M o o r e et al 1 9 6 2 ) f o u n d t h a t t h e p H optimum for starch digestion was 6.8. Undoubtedly, the pH optimum for some of the other activities carried on by rumen microorganisms may vary from these two points. N e v e r t h e l e s s , it i t c a n b e s a f e l y s a id id t h a t m o s t o f the. rumen microbial activity occurs at an optimum rate between a pH range of 6.7 and 7.0. The necessity for adjusting pH depends largely upon the rate, extent an d type of ferm e n t a t i o n . D u r i n g t h e c o u r s e o f c e l lu lu l o s e d i gestion acid production occurs at a much slower rate than during starch digestion. As a

of a somew hat more complicated apparatus , since it is most successfully performed with i n d i v id i d u a l t u b e s l e a d in in g f r o m a c o m m o n m a n i f o ld l d t o e a c h f e r m e n t a t i o n v e s se se l . T h e B u n s e n v a l v e t e ch ch n i q u e d e p e n d s o n t h e a b i l i t y o f t h e fermentation to produce enough CO2 during the phases of metabo lism to keep the atm os p h e r e a n d t h e li l i q u id id m e d i u m s a t u r a t e d . I n 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 resatu rated with CO2 b y f a i r l y v io io l e n t b u b b l i n g a f t e r e a c h s a m p l in i n g . T h i s v i o l e n t a g it it a t i o n m a y h a v e a n effect on the fe rme ntation for a short period of time after agitation. Temperature The temperature usually sel e c t e d f o r i n vi t ro f e r m e n t a t i o n s h a s b e e n 3 9 ~ C., although occasionally others are reported. A l t h ou ou g h c o n s t a n t t e m p e r a t u r e w a t e r b a t h s a r e g e n e r a l ly ly p r e f e r r e d a s t h e m e a n s o f m a i n t a i n in i n g t e m p e r a t u r e , s o m e i n v e s ti ti g a t o r s h a v e used incubators for this purpose. Either method is satisfactory as long as a reasonably constant temperature is maintained. However, t e m p e r a t u r e s h o u l d d e f i n i t e ly ly b e s t a n d a r d i z e d , s i n c e d i f f e r e n c e s o f a s l i t t l e a s 0 .5 ~ C. m a y invalidate comparisons between individual fermentations. In addition, care should be taken to prevent the temperature from rising

consequence, wh en the buffering m ediu m described earlier is used, the pH 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 s t u di di e d , h o w e v e r , t h e p H m a y d r o p m u c h more rapidly due to the rapid rate of fermentation of this more soluble type of carbohydrate. In studies on adapting all-glass fermenta tio n sy ste ms to sta rc h d ig e stio n , M o o re et al 1962) found that the pH did not drop m a r k e d l y u n t il il b e t w e e n 6 a n d 1 2 h r . a f t e r inoculation. After the fermentation started, however, the pH dropped very rapidly, and f o r m a x i m u m a c t i v i ty ty i t w a s n e c e s s a r y h o u r l y to readjust the pH 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, pH changes may commence during the first hour after inoculation, and the drop in pH may be much more rapid. When a rapidly hydrolyzed source of ammonia nitrogen such as urea is being used as a n i t r o g e n s o u r c e , t h e e f f ec ec t o f a c i d p r o d u c t i o n on pH is modified. In some cases where amm o n i a p r o d u c t i o n i s v e r y r a p i d a n d a c id id p r o duction has not commenced, the pH may actually rise above 7. This is usually quite t e m p o r a r y b u t m a y b e d e l e t er e r i o us u s , s i n ce ce t h e rumen bacteria seem to be somewhat more

ot ivoenr. 4R0 u~mCe.nd ub raicntge r ai al l apphpaes ae rs ot of tbhee ef se pr m e ceina tl al ysensitive to high temperatures and, if exposed

s e nGs ei tni ev rea lt ol y ,p Hp Hl e vi es l saadbj uo svtee dt h af rno m b e l tohwe 7a. c i d side to 6.9 with a solution of sodium carbonate

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FO R R U M E N

S T U D IE S

861

( s a t u r a t e d ) a n d a d j u s t e d f r o m t h e b a s i c s id id e to 6.9 with a phosphoric acid solution. Other acidic and basic solutions could possibly be u s e d to t o a d j u s t t h e p H ; h o w e v e r , t he h e s e t w o co co r r e s p o n d t o t h e u s u a l b u f f er er i n g s y s t e m e m ployed in the media. Inoculum Sources. T h e s o u r c e o f i n o c u l u m

o n t h e s a m e r a t i o n a n d c o m p o s i t e t h e i n o c u lu lu m d r a w n f r o m a ll ll f o u r a n i m a l s o n a n y g i v e n d a y of study. By so doing the chance of one anim a l ' s b e i n g s l i g ht ht l y o f f f e e d a n d t h u s g r o s s l y a f f e c t i n g t h e in vitro r e s u l t s w o u l d b e m i n i mized. It has been our experience that it is e a s i e r t o d e t e c t a n a n i m a l t h a t i s off f e e d

f o r in vitro r u m e n f e r m e n t a t i o n s a l s o r e p r e sents the source of the greatest possible error or varia bility as well as the po ssibility for misinterpretation of the results. The two major c o n s i d e r a t i o n s a r e ( a ) t h e c h o i c e o f th th e s o u r c e animal or animals and (b) the methods of inoculum preparation. These will be considered in the order given. Several investigators (W arne r, 1956; Bow ie, 1962) hav e pointed out the importance of utilizing a source animal, which is being maintained on a ration s i m i l a r t o t h a t b e i n g s t u d i e d in vitro. T h i s i s especially true for those using the continuous f lo lo w o r c h e m o s t a t t e c h n i q u e s . A l t h o u g h t h e rumen has the advantag e of abounding in a variety of species and types of bacteria and protozoa, it also has the concomitant disadv a n t a g e o f t h e a b i l i t y o f t h e se se g r o u p s o f m i c r o organisms to change in proportion to each o t h e r v e r y r a p i d l y . M a n y t i m e s th t h e s e c h a n g es es 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 d i g e s te t e r s , p r o t e o l y t i c , e t c .,., i t i s n o t t e c h n i c a l l y 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 fro m 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 s t u d i e d t h e u s e o f in vitro f e r m e n t a t i o n s f o r t h e e v a l u a t i o n o f f o r a g es e s . M o s t o f t h e se se w o r k ers 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 espec i a l ly l y i m p o r t a n t , w h e n o n e is is a t t e m p t i n g t o com pare quantitative ly the data for more than

by studying its inoculum in the laboratory than by observing the animal in the feeding pen. W h e n d e t e r m i n i n g f o r a g e d i g e s t i o n in vitro some workers have observed that inocula taken from animals fed alfalfa hay were sup e r i o r t o i n oc o c u l a t a k e n f r o m a n i m a l s f e d g r a ss ss 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. M e t h o d s o f i n o cu cu l u m p r e p a r a t i o n v a r y f r o m the simplest technique of using whole rumen fluid taken directly from the animal's rumen to the use of was hed cell suspensions and en-

oMnoeo rdea yet. al. I n (t1h9e 6 2s)t u df oyu nodf i ts t ac rocnhv e nd ii eg ne ts tai on nd advisable to use four inoculum-source animals

tphr ee s sr u m a ne dn ct ho en t efni trss t wleirqeu i sdq ueexetzreadc t i nw aas l adrigs ecarded. The squeezed pulp was then resus-

r i c h m e n t c u l tu t u r e s . A g a i n t h e c h o ic ic e o f p r e p a ration techniques depends on the phase of r u m e n a c t i v i t y b e i n g s t u d ie ie d . W h o l e r u m e n f lu l u id id m a y b e r e m o v e d f r o m t h e r u m e n b y t w o b a s i c t e c h n iq iq u e s . T h e f i r s t is by a spiratin g or drawing off a sam ple 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 i s s i m p l y t o r e m o v e a s u f f ic ic i e n t q u a n t i t y o f rumen contents b y hand, place it in a press a n d s q u e ez e z e t h e l iq iq u i d f r o m t h e m a t e r i a l . T h i s t y p e o f i n o c u l u m i s i n v a r i a b l y f i lt l t e re re d t h r o u g h several layers of cheesecloth. It 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 grow th factors for the microorganisms. If the object o f t h e s t u d y i s to to m e a s u r e t h e r e q u i r e m e n t 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. It also represents the fastest means of transferring microorganisms from t h e r u m e n t o t h e in vitro f e r m e n t a t i o n f l a s k s . A modification of this technique was dev e l o p e d b y J o h n s o n et al. ( 1 9 5 8 ) , i n w h i c h

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pended in a pH 7 phosp hate buffer, agitated and re-squeezed. This second extract was then used as the inoculum and has become known a s t h e p h o s p h a t e b u f fe fe r e x t r a c t . T h i s p r o c e d u r e w a s d e v e l o p e d w i t h t h e f a c t in in m i n d t h a t m a n y o f t h e c e l l u l o l y t ic ic o r g a n i s m s e x i s t i n c l o s e a s s o c i a t i o n wi t h t h e f i b r o u s p o r t i o n s 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. Th is techniqu e would not necessarily be advisable when one is studying digestion of starch or other ration constituents, however. More refined inoculum preparation techn i q u e s d e p e n d u s u a l ly ly o n v a r i o u s m e a n s o f 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 b y M c N a u g h t ( 1 9 5 1 ) a n d l a t e r to to a g r e a te te r e x t e n t b y B e n t l e y et al. ( 1 9 5 4 a , b , 1 9 5 5 ) . The latter workers found it feasible to use a Sharples supercentrifuge to concentrate the bacteria in batch form to prepare the inocul u re re . A l t h o u g h t h e i n o c u l u m d e v e l o p e d b y Johnson et al. 19 58 ) was almost completely inactivated by two washings with phosphate b u f f e r , C h e n g et al. ( 1 9 5 5 ) d e v e l o p e d a p r o c e d u r e u s i n g wa s h e d c e l l s u s p e n s i o n s , wh i c h was used to study nutrient requirements of t u r e e n mi c r o o r g a n i s ms b y Ha l l et al. ( 1 9 5 3 ) a n d Hu b b e r t et al. 195 8a, b). Since then several workers have used washed cell suspensions successfully in in vitro rumen fermentat i o n s . D e h o r i t y et al. ( 1 9 6 0 ) p u r i f i e d t h e c e l l u lo lo l y t i c i n o c u l u m e v e n f u r t h e r b y s e p a r a t ing the fraction sedimented between 1,500 and 3,000 g. In studying starch digestion M o o r e et al. ( 1 9 6 2 ) c e n t r i f u g e d t h e t u r e e n bacteria at 3,000 g and resuspended this sedim e n t f o r a n i n o c u lu lu m . 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 bu t steady l o ss ss i n c e l l u l o l y t ic ic a c t i v i t y b y i n c u b a t i o n a n d a e r a t i o n w a s d e m o n s t r a t e d b y J o h n s o n et al. (1958). Nevertheless, these techniques may be necessary in order to free the bacterial preparation of endogenous nutrients and gr er qo uwitrhe mfeanctt o rfso.r Fsohro r et -xcahma pi nl e , f aat t ym oarcei d es x abcyt m i c r o o ga g a n is is m s w a s d e m o n s t r a t e d b y D e h o r i t y

ti c b a c t e r i a 1 9 6 0 ) b y g r o w i n g c e l l u l o l y ti in an in vitro system for a period of time and et al.

then reharvesting the cells as an inoculum for future fermentations. By so doing blanks givi n g z e r o c e l l u l o s e d i g e s t i o n we r e o b t a i n e d . F e r m e n t a t i o n T i m e P e r i o d s. s. T h e t i m e o f 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 s t u d i es e s i n t h e p a s t . A f ew ew b a s i c c o m m e n t s 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 m atter of m i n u t e s a f t e r c o m b i n in i n g w i t h t h e i n o c u lu lu m and often peak ac tivity 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 a f t e r 2 4 h r . ( M o o r e et al. , 1 9 6 2 ; e l - S h a z l y et al. , 1 9 6 1 a ) . H i g h l y s o l u b l e s t a r c h e s w i l l b e 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 betw een the digestion of cellulose in its native form in i oing eosft i op nu r ioffi enda tcievlel u lfoosresf ou rcahg eass aSnodl ktah eF dl oi gc .e4s tD age celluloses appears to be initiated at a mu c h e a r l i e r t i me , i . e . , d i g e s t i o n i s d e t e c t a b l e 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 differe n c e s a r e n o t k n o w n a s y e t . B a k e r et al. ( i 9 5 9 ) h a v e s h o w n t h a t t h e r a t e o f d i g e s ti ti o n o f p u r i f i e d c e l l u l o s e d e c r e a s e s wi t h i n c r e a s i n g degrees of crystallinity. They further suggest t h a t t h e n a t i v e c e l l u l o s e i n f o r a g e s i s mo s t l y 9 The Solka Floc ment ioned 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 n v tro tureen fermentations. It should be kept in mind, however, that there are several grades grades a4~d a4~d types of Solka Floc produced by this company; the one to which we refer refer in this paper is Solka Floc BW-40.

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PROCEDURES FOR RU M EN in the am orphou s 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 p h y s i c a l p r o p e r t i e s i s d e s ir ir a b l e . A further consideration should be made when the objective involves measurement of r a t e p h e n o m e n a . I n m o s t c l os os e d s y s t e m s i t is impossible to provide sufficient substrate t o m a i n t a in i n t h e m a x i m u m r a t e o f f e rm rm e n t a tion for long periods. In o ther w ords the sub strate 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 d i g e st s t i on on i s m a n d a t o r y . T e r m i n a t i n g t h e F e r m e n t a t io io n . P r a c t i c a l l y a n y m e a n s o f s t o p p in in g e n z y m a t i c a c t i v i t y c a n be used to terminate in vitro rumen fermentations. In some cases it suffices merely to rem o v e t h e t u b e s f r o m a w a t e r b a t h a n d r e f r ig ig erate them. Fo r mo re 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 portion s of the substrate material. Assuming it does not interfere with subsequent analytical procedures, the method of choice used in our l a b o r a t o r y is i s th th e a d d i t i o n o f a p p r o x i m a t e l y 1 ml. of saturate d m ercuric chloride solution per 100 ml. of fermentation volume. Analytical Procedures. No attempt will be made to elucidate the numerous analytical

STUD IES

863

Interpretation of In Vitro R e su l t s

In a discussion of the interpretation of in vitro rumen fermentation data, one can only enum erate points of caution that should be considered. Actual methods of interpretation or analysis of data must be left to the inditvoi d rueailt ei rnavtees tai gn a toobr .j eFc it ri sotnl y t, ot hth teh ea uttehromr c haorot isfeisc i a l r u m e n , s i n c e t h e c lo lo s e d s y s t e m s u s e d are generally in no way intended to duplicate exactly wh at is happening in the rumen. Th e term in vitro rumen fermentation is pref e rr rr e d . C r i t e r i a f o r v a l i d i t y f o r s u c h f e r m e n tations have been published by Warner ( 1 9 56 56 ) a n d D a v e y e t a l . ( 1 9 6 0 ) . I n t h e d e sign 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 a s p o s s i b le le . N e v e r t h e l e s s , c l o s e d s y s t e m p r o cedures are often usable for studying specific phases of rumen microbial metabolism, and in doing so one or more criteria may have to

procedures that might be used in following the activity of in vitro rumen fermentations. For m ost purposes procedures that a re useful f o r t h e p a r t i c u l a r m a t e r i a l s b e i n g s tu tu d i e d c a n be modified to be suitable for analyzing in vitro rumen fermentations. It must be kept in mind in interpreting results, however, that w h i l e s u b s t r a t e m a y b e d is is a p p e a r i n g d u r i n g the fermentation, a large quantity of bacterial cells is being produced--a fact which m a y c o m p l i c a t e t h e t e r m i n a l a n a l y s is is . F o r example, studies on the proteolysis of natural feed proteins have had to be restricted to i n d i r e c t m e a s u r e m e n t s o f p ro ro t e o l y s i s s u c h a s ammonia formation, since there is no easy

be sacrificed to accomplish the task. Consequently, the criteria which have been sacrificed must be kept in mind when interpreting the results. It 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. T h e r e f o r e , t r a n s p o s i t i o n o f co co n c l u s i o n s i s n o t 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 c o m p a r e d d i r e c t ly ly w i t h n a t u r a l a n i m a l r a tions and the processes within the rumen. T h e c h e m i c a l n a t u r e o f n a t u r a l f e e ds d s t u ff ff s i s indeed a com plex 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. It is of imm ediate 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 r u m i n a n t s ( P i g d e n a n d B e ll l l , 1 9 55 55 ; K a m s t r a

w b ei ct w p raoyt e ti on d ai sntdi n g tuhi es h m r oebeina l t h ep r on taet iunr a l b feei ne dg synthesized.

ee tt aa ll .. ,, 11 99 55 89 ;; Q D uo incekfee r e te ta la. l, . , 1 19 95 96 ;0 ; HBe ar suhmbge ar gr de tr e t a l . , 1 9 6 2 a , b , 1 9 6 4 ; Re i d e t a l . , 1 9 6 4 ;

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864

JOHNSON

J o h n s o n et al. 1962a, b, 1964; Tilley et al. 1960; Tilley and T erry , 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 s e e m t o b e s o m e in i n c o n s i s t e n c y in in t h e i n t e r p r e t a t io i o n o f t h is is t y p e o f i n f o r m a t i o n . B e c a u s e o f t h e i n te te r e s t s h o w n b y w o r k e r s i n m a n y o t h e r 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 in take figures. Th e 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 i n v i v o dat a from t he 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 ch aracteristics of forages differ gre atly 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 interpretat io i o n o f t h e d a t a . M a n y s p e ci c i es e s o f f o r a g e s f o rm rm a uniform pattern within species in their relationship between in vitro and i n v i v o data. Indeed some species can be grouped together and still form highly significant patterns.

to this variation, if each of the laboratories were calculating regression equations from their in vitro data and were using standard i n v i v o data, widely different regression lines would be obtained. In addition to this, a collaborative i n v i v o digestion trial E. Do nefe r, u n p u b l i s h e d d a t a , M a c D o n a l d C o l le le g e , Q u e bec, Canada) demonstrated that considerable variation occurred also in the conduct and r e s u l t s o f i n v i v o t r ia ia l s, s, e v e n w h e n a s t a n d a r d 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 r e g r es e s s io io n e q u a t i o n s d e v e l o p e d i n o t h e r l a b o r a tories, even though identical techniques may b e i n u s e in in b o t h l a b o r a t o r ie ie s . I t h a s a p p e a r e d to be convenient and popular, especially in some other countries, to utilize in vitro rum en fermentations to study a series of forages and t h e i r d i g e s t ib i b i l it it y w i t h o u t a c c o m p a n y i n g i n v i v o digestion trials to establish proper regression equations. Indeed, this is the ultimate goal of those working with the techn i q u es e s . H o w e v e r , t h e v a l i d i t y o f t h e r e g r e s s io io n 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 o n e i s s t u d y i n g t r o p i c a l o r s e m i t r o p i c a l g r as as s e s . T h e 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 a nd

aNme vo en rgt hseol emses , s pdeecf ii ensi t eh a va ne d b egerno sos b sdeirfvf ee rde nacneds have necessitated the use of considerable caution in interpreting results. For example, J o h n s o n et al. 1962b) observed t hat a di fference 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 h a s r e c e n t l y b e e n c o m p l e t e d in in w h i c h 1 7 laboratories used various in vitro fermentation techniques to study the digestibility of s t a n d a r d f o r a g es es B a r n e s , 1 9 6 6 ) . T h i s s t u d y

rt eh ge r ei sn s i voint r oe qruuam t ieonn s f ehramveen tbaet ieonn dm e taeyr mpi rnoevde, highly valuable in studying forages. Generally, data can be obtained by many of the techniques which are highly correlated with d r y m a t t e r d i g e s ti t i b il i l it it y . T h i s h a s b e e n r e a d i l y s ho ho w n b y B a u m g a r d t e t a l . 1 9 6 2 a , b , 1 9 6 4 ) , Johnson et al. 1 9 6 4 ) , H e r s h b e r g e r et al. 1959), Rei d et al. 1 9 6 4 ) a n d T i l l e y et al. 1960). An excellent application of these techniques to the measurement of herbage digestibility and intake by grazing livestock h a s b e e n p u b l is is h e d b y V a n D y n e 1963 ), V a n D y n e a n d M e y e r 1 9 6 4 ) an an d V a n D y n e and W eir 1964).

demonstrated that considerable variability occurred within and between the techniques e m p l o y e d a t t h e d i f f e r e n t l a b o r a to to r i e s . D u e

Rumen Protozoology

When the large populations of protozoa

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PROCEDURES

FOR RUM EN STUDIES

p o s s ib ib l e i n r u m e n f e r m e n t a t i o n s a r e c o n s i d e r e d, d , i t is is u n f o r t u n a t e t h a t t h e i r r o le le i n o v e r a l l m e t a b o l i s m h a s n o t b e e n m o r e f u l l y s tu tu d ied. 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 v itr o . A l t h o u g h s e v e r a l w o r k e r s h a v e s u c ceeded in keeping rumen protozoa alive for s e v e r a l w e e k s in v itr o , t o t h e b e s t o f t h e autho r s knowledge none of these preparations has been completely free of bacteria. T he close association of some protozoa and rumen bacteria thus makes interpretation of metabolic data very difficult. No review of t h e t e c h n i q u es e s f o r s tu tu d y o f r u m e n p r o t o z o a w i l l b e g i v e n h e r e , b u t t h e r e a d e r i s r e f e r re re d t o t h e f o l lo lo w i n g r e fe fe r e n c e s ( C l a r k e , 1 9 6 3 ; Colem an, 1 9 6 0, 0, 1 9 6 2 ; Eadie, 1962a, b; G u t i e r r e z , 1 9 55 5 5 ; G u d e r r e z a n d D a v i s , 1 9 59 59 , 1 9 6 2 ; H u n g a t e , 1 9 5 5 ; H u n g a t e et al., 1964; M a l l , 1 96 9 6 4 ; M a h a n d H u n g a t e , 1 9 6 5; 5; O x f o r d , 1 9 5 5 ; Q u i n n et al., 1 9 6 2 ; W a r n e r , 1 9 6 2 ) . Pure Culture Techniques T h e s t u d y o f r u m e n m i c r oo o o r g a n is is m s i n p u r e 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 b e a s u b j e c t f o r a c o m p l e t e r e v i e w b y i t s el el f . For further detail the reader is referred to Bry ant (1959), Hun gate (1950) and Hun gate et al. (1964).

865

al. ( 1 9 6 2 ) . B a s i c a l ly l y , t h e a p p a r a t u s c o n s i st st s 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 f i s tu tu l a t e d a n i m a l . T h e f e r m e n t a t i o n w i t h i n 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 i n e q u i l ib ib r i u m w i t h t h e e n v i r o n m e n t o f t h e r u men 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 inocu l u m i n th th e s a m e m a n n e r a s a n i n v i t r o fermentation. P e t t y j o h n et al. ( 1 9 6 4 ) d e s c r i b e d a d i f f e r ent 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

The VIVAR technique refers specifically to

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 t i g h t l y t ie i e d ( f ig ig u r e 2 ) . T h e s e b a g s a r e t h e n p l a c e d i n t h e t u r e e n o f a f i s tu tu l a t e d a n i m a l b y a v a r i e t y o f te te c h n i q u e s a n d r e m o v e d a f t e r v a r i ous periods of time to determine digestion of t h e c o n t en e n t s . P r e c a u t i o n s m u s t b e t a k e n t o i nns u r e a m e s h s u f f i c i e n tl tl y f i n e t h a t p a r t i c l e s o f 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 rum e n . H o p s o n e t a l. ( 1 9 6 3 ) c o m p a r e d t h i s t e c h n i q u e w i t h d e t e r m i n a t i o n o f d ig i g e s t i b il i l i ty ty b y i n vitro procedures. Generally, the coefficients of variation for the dacron bag technique were v e r y h i g h f o r d i g e s ti t i b i li li t y v a l u e s a t e a r l y t i m e periods from 6 to 24 hr. The digestion curves

an in vivo artificial rumen apparatus origin a l l y d e s c r i b e d b y F i n a et al. ( 1 9 5 8 ) a n d l a t e r m o d i f i ed ed b y T e r e s a ( 1 9 5 9 ) a n d F i n a e t

o b t a i n e d f r o m u s i n g t h e s e t e c h n iq iq u e s , h o w ever, were similar to digestion curves obtained u s i n g in v itr o f e r m e n t a t i o n s . I t w a s a l s o n o t e -

In Vivo Techniques A l t h o u g h n o a t t e m p t w i ll ll b e m a d e i n t h i s r e view to give a complete coverage of in vivo techniques for investigating rumen function, i t is i s f e lt lt t h a t s o m e m e n t i o n s h o u l d b e m a d e o f a number of them, since they bear directly on t h e s t u d i e s o f th th e f u n c t i o n o f r u m e n m i c r o o r ganisms. The discussion will be restricted to techniques other than digestion trial techniques, since these are discussed in ~nother chapter of this monograph. VIVAR Techniques

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866

JOHNSON Rate

o / P a s sa sa g e M e a s u r e m e n t s

Al t h o u g h n o t a d i r e c t m e a s u r e o f r u m e n 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

Figure 2. Nylon bag shown with nylon sus p e n si o n c o r d a n d i d e n ti fy i n g ta g .

of the process rumen Since digestion, especially in reference to of forages. a considerable portion of ruminant rations may be indigestible, in some investigations rate of passage c o u ld ld c o n c e i v a b l y b e a m o r e i m p o r t a n t m e a s u r e m e n t t h a n d i g e s t i b i l i t y . Re i d ( 1 9 6 1 ) h a s estimated that 90 of the variation in total e n e r g y i n t a k e b e t we e n f o r a g e s c a n b e a c c o u n t e d f o r b y v a r i a t i o n s i n t h e i n t a k e . Vo l u n tary 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) a n d Ca s t l e ( 1 9 5 6 ) , i n wh i c h p o r t i o n s o f t h e r a t i o n a r e s t a i n e d wi t h a p a r t i c u l a r d y e a n d

wo e tahniinm a rl t hoyn t dh iagt eas nt i beiflfiet yc t ooff tthhee rsautbi sotnr aot ef st hwi t h e b a g s wa s n o te te d . Al t h o u g h t h e v a r i a b i l i t y a n d t h e v a lu l u e s f r o m s u c h t e c h n iq iq u e s a p p e a r to be high, the dacron bag technique as well a s t h e VI VAR t e c h n i q u e a p p e a r s t o h a v e some application in determining the effect of v a r i o u s r a t i o n t r e a t m e n t s o n d i g e s t i o n wi t h i n the rumen. It 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. ( 1 9 6 1 a ) , Va n Keur en and Hei nem ann ( 1962) , Bur t on et al. ( 1 9 6 4 ) , Be l a s c o et al. ( 1 9 5 8 ) , E r wi n a n d Elliston (1959), Miles (1951), Quin et al. ( 1 9 3 8 ) , M c An a l l y ( 1 9 4 2, 2, 1 9 4 3 ) , Ba l c h a n d

the number of stained particles excreted in the feces over a period of time a re coun ted visually. Equations for calculation of mean r e t e n t i o n t i m e we r e d e s c r i b e d b y Ca s t l e (1956). In the determination a cumulative excretion curve is drawn, and the times for e x c r e ti ti o n o f 5 to 95 (at 10 i n t e r v a ls ls ) of the total particles excreted are determined from the curve. These times are summed and divided by 10 to determine R or mean r e t e n t i o n t i m e . T h e s e t e c h n i q u es es a r e e x t r e m e l y laborious and at best are only estimates of r a t e o f p as as s a g e . Ca r e m u s t b e t a k e n t o i n su su r e that the stained material resembles physically the component of the ration that is being studied.

J ouhsnks oetn al.( 1 9( 15906) ,2 ) .Ar c h i b a l d et al. ( 1 9 6 1 ) a n d L Although similar in principle, a considerably different technique mechanically is the use of suspended cotton threads in the rumen, ( Ho f l u n d et al., 1 9 4 8; 8; Ba l c h a n d J o h n s o n , 1950; Campling et al., 1 9 6 1 ) . T h i s t e c h n i q u e has been used to study rumen microbial activity involving cellulose digestion. In this c a s e t h e t h r e a d s a r e we i g h e d a n d s u s p e n d e d in the rumen for a period of time, after which t h e y a r e wi t h d r a w n a n d w a s h e d t h o r o u g h l y . Af t e r d r y i n g t h e y a r e we i g h e d a g a i n a n d t h e activity is determined by loss of weight. Aust r a l i a n wo r k e r s h a v e r e c e n t l y s h o wn t h a t a n alysis of the coils of thread for nitrogen after

A modification of the use of stained feed particles is the use of inert plastic particles. T h e s e c a n b e m a n u f a c t u r e d wi t h d i f f e r e n t size, color and density and thus present an a r r a y o f c h a r a c t e r i s t i c s wh i c h m a y b e t e s t e d . Ki n g a n d M o o r e ( 1 9 5 8 ) c o m p a r e d th th e p a s sage of plastic particles of different size and density. Although the use of stained particles is r e c o m m e n d e d wh e n r a t i o n s o f d i f f e r e n t t e x ture 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. S u c h a t e c h n i q u e wa s e m p l o y e d i n o u r l a b o r a -

periods of suspension in the rumen yields data which also correlate well with microbial act i v i t y ( R. J . M o i r , p e r s o n a l c o m m u n i c a t i o n ) .

ot of rbyo t(hu nr pouubglhias gh ee da ndda tcao) n. cTe nh ter artaet er a ot ifo pn as s s( ba gu et wi t h a l l a n i m a l s g e t t i n g t h e s a m e r a t i o n d u r -

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PROCED URES

FO R R U M E N

S T U D IE IE S

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(1928), and since that time numerous m odifications of the fistulation techniques and design of cannulas have been developed. The procedure described by Schalk and Amadon (1928) was essen tially a one-stage surgical technique, while the more recent procedure d e s c r i b e d b y J a r r e t t ( 1 9 4 8 ) c o n s i s t s o f a tw tw o s t a g e o p e ra ra t i o n . B o t h t e c h n i q u e s h a v e b e e n widely used. Since the original publications b y S c h a l k a n d A m a d o n ( 1 9 28 28 ) a n d J a r r e t t ( 1 9 4 8 ) m a y n o t b e r e a d il i l y a v a i la l a b l e to to t h e student desiring to know more about fistulation procedures, a more detailed description of these procedures will be given. The descript i o n s w i l l in in c l u d e m o d i f i c a t i o n s a n d t e c h n i q u e s used in the author's laboratory and do not

Th e 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 b e e n t r a n q u i l i z e d , t h e l e f t f la la n k r e g i o n b e t w e e n t h e l a s t r ib ib a n d t h e h i p b o n e i s d i p p e d , s h a v e d , scrubbed with an antiseptic soap and painted with a tincture of iodine solution. The line o f in i n c i s i on on i s a n e s t h e t i z e d l o c a l l y w i t h a 2 procaine solution by injecting subcutaneously and intraderm ally. 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 s u f f ic i c i e n t ly ly a n e s t h e t i z e d , a v e r t i c a l i n c i s i o n i s 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 by 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 i s d r a w n t h r o u g h t h e c l a m p , w h i c h is is t h e n closed by turning the nuts. It 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 s e e m d e s i r a b l e f o r t h e l e n g t h o f i n c i s io io n i n volved. The area of the wound may then be t r e a t e d t o p i c a l l y w i t h a n t i b i o t ic ic m a t e r i a l s a n d s p r a y e d w i t h p l a st st i c b a n d a g e . T h e s u tures ma y be removed after 7 to 9 days, at which time the necrotic section of the rumen wall may be excised and the clamps removed. It may be desirable merely to wait until the necrotic section has dropped away prior to subsequent treatment. It may also be necess a r y a t t h a t t i m e t o t r e a t m i n o r l e si s i o ns n s s t il il l

necessarily reflect the exact description published b y the original authors. Schalk and Am adon Procedure Figure 3).

remaining along the incision line prior to inserting the cannula in the fistula. As soon as the swelling has subsided and healing is

i n g t h e s a m e p e r i o d ) w a s s t u d i ed ed b y d o s i n g the animals with chromic oxide in capsules. Feces were collected at various intervals over a p e ri r i o d o f t im im e a n d t h e n a n a l y z e d f o r c h r o m i c 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 ( C h a n d l e r et al., 1 9 6 4 ) . I t i s t h e o p i n i o n o f the author that considerably mo re work should be conducted on techniques for measuring rates of passag e of feed residues in ruminant animals.

Rumen Fistulation Techniques Although the presence of several distinct anatomical portions in the digestive tract of t h e r u m i n a n t m a y b e b e n e fi f i c ia i a l a s fa fa r a s t h e animal's welfare is concerned, it complicates t h e s t u d y o f t h e d i g e s t iv iv e p r o c e s s a n d t h e rt raat ec t . o fI n f e er de c epnat s s aygeea r st h rsouurgghi c atlh e t e dc ihgnei sqtui ve se have been developed and refined to the point that direct access to practically any portion of the digestive tract is possible. It is further possible not only to obtain samples from these various portio ns of the digestive tract, but also to recycle the contents back into the tract after sampling or after making certain a d d i t i o n s a t s p e c i f i ed ed p o i n t s a l o n g t h e t r a c t , 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. R u m e n f i s tu t u l a t io i o n t e c h n i q u es es w e r e d e s c r i b e d many years ago by Schalk and Amadon

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F i g ur e 3 . Se v e r a l s t a g e s o f Sc ha l k a nd Am a do n 1 9 2 8) 8) f i s t ul a t i o n pr o c e d u re re : A ) i n c i s i o n w i t h r u m e n f o l d p u l l e d t h r o u gh gh , B ) p l a c i n g w o o d e n c l a m p o n r u m e n f o ld ld , C ) i n s e r t i n g s u tu t u r e s a l o n g c l a m p e d a re re a, a, an an d D ) t y i ng s ut ur e s .

nearly complete a cannula should be inserted to close off the ru men . It is impossible to obtain a fit sufficient to

divided by blunt dissection. If there is a heavy f a t d e p o si si t i t m a y b e m o r e c o n v e n i e n t t o remove some of the fatty tissue. Blunt dissec-

pcedure r e v e n t especially l e a k a g e o f with r u m elarger f l u i dopenings. wi t h t h i sHo p rwolnarger e v e r s o m e e x p e r ie i e n c e m u s t b e g a in in e d b y t h e operator before the proper selection of the size of incision and rumen fold can be made to allow for the desired fistul fistulaa opening. Jarrett Procedur Proceduree Figure 4). F o r t h i s p r o cedure the animal is similarly fasted and tranquilized. The area of surgery is cleaned and anesthetize d locally with pro caine solution. T h e s u r g ic ic a l i n ci ci s io io n i s m a d e m i d wa y b e t we e n the last last rib and the hip joint starting app roximately 2.5 cm. below the transverse processes. It is continued ven trally for about 7 to 10 cm. for sheep destined to be fitted with the origin a l J a r r e t t c a n n u l a . T h e t wo e d g e s o f t h e i n -

tion techniques are used to divide the three distinct layers of muscle fibers lying between the surface and the peritoneum. After the initial dissect dissection ion the muscle layers are then separated by means of the fingers to give access to the peritoneum. When the peritoneum is reached it should should be incise incisedd in such a wa y that the two edges of the incision can be p i c k e d u p a n d h e l d wi t h h e m o s t a t s . T h i s i n cision 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

cision are folded back by the attachment of hemostats placed along the edge. The superficial fascia which lies immediately below is

the exteriorized rumen pocket by picking up t h e m u s c u l a r wa l l o f t h e r u m e n wi t h t h e s u turing needle continued throug h the perito-

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Fi g u r e 4 . Fi r st sta g e o f J a r r ett ett 1 9 4 8 ) fi stu l a tio tio n p r o c e d u re re : A ) e x p o se d h e r n i a i n r u me n w a l l b e i n g se c u r e d to sk i n b y sta y su tu r e s, B ) ty i n g th e sta y su tu r e s o v e r g a u z e r o l l , C ) s ta y su tu r e s c o mp l e te d sh o w i n g e x p o se d h e r n i a p r i o r to c l o si n g i n c i sio sio n , a n d D ) c o mp l e te d fi r st sta g e .

neum and brought externally through the skin approximately 2.5 to 5.0 cm. from the

about 1 cm. of the original incision opening is left open to assure a drainage channel. The

incision a r e a v o i dline. e d s oThe t h a t muscle t h e s u tlayers u r e s r e and t r a c t fascia them from the herniated area. When both ends of the suture silk have been broug ht 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

incision line t r e a t e d wi t h aand n t i b ipoints o t i c o i noft m stay e n t a sutures n d s p r a yare ed wi t h p l a s t i c b a n d a g e . Af t e r 1 0 d a y s t h e s t a y 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 a f t e r t h e i n i t ia ia l o p e r a t i o n , b u t c a n b e p e r formed 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 t h e h e r n i a c l e a n e d a n d a n e s t h e t i z e d wi t h p r o caine. An incision is made in the hernia di-

with a continuous line suture. Care should be t a k e n d u r i n g c l o s u r e t o i n s u r e t h a t t h e t wo edges of the incision are everted and that

r e c t l y t h r o u g h t h e s k i n a n d t h e r u m e n wa l l . This incision should be approximately 2.5 cm. long for fitting the original Jarrett can-

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nula and should be started as close to the dorsal border of the hernia as possible. It may be convenient, but not necessary, for insertion of the cannula to use retractors to hold the two surfaces of the incision apart a n d i n a n u p wa r d a n d o u t wa r d d i r e c t i o n . Th e cannula figure 5) is 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 s m a l l p r o t r u s i o n t h a t m a y b e in i n s e r t ed ed t h r o u g h 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 c a n u s u a l l y b e wo r k e d o u t c o m p l e t e l y i n t o a n u n f o l d e d o r u n k i n k e d f l a t s u r f a c e wi t h i n the rumen. The area around the fistula openi n g s h o u l d n o w b e c l e a n e d a n d t r e a t e d wi t h

d e s i g n d e s c r i b e d b y He n t s c h l et al 1954) is convenient for use with large openings such as are usually provided in bovines by the S c h a l k a n d Am a d o n 1 9 2 8 ) p r o c e d u re re . J a r r e t t 1 9 4 8 ) d e s c r i b e d a r u b b e r c a n n u l a wh i c h is very economical and suitable for use with his surgical procedure. The original Jarrett c a n n u l a 5 h a s a n i n si si d e d i a m e t e r o f a p p r o x i m a t e l y 2 6 m m . f i g ur ur e 5 ) . A c a n n u l a v e r y similar in design, but somewhat larger in inside diam eter, i.e i.e., ., 38 ram., is also availab le 6 a n d m a y b e m o r e s u i t a b le le f o r p r o c e d u r e s r e quiring 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.

aient n a ntot i binstall i o t i c p the r e p a steel r a t i o ncollar . I t i s onm othe s t coutside onvenby 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 c o l l a r m a y t h e n b e wo r k e d d o wn a r o u n d t h e pliers over the neck to the proper depth. T h e m o u t h o f t h e c a n n u l a i s t h e n s e a l e d wi t h 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. It 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.

u s e f u l i n s t u d y i n g i n t a k e a n d d i g e s t ib ib i l i ty ty o f grazed forages. Many of the basic techniques a n d d e s ig i g n s we r e d e v e l o p e d b y T o r e l l 1 9 5 4 ) , B a t h et al 1 9 5 6 ) , Co o k et al 1 9 5 8 ) , L e s p e r a n c e et al 1 9 6 0 ) , M c M a n u s 1 9 6 1 , 1 9 6 2) 2) a n d M c M a n u s et al 1 9 6 2 ) . On e o f t h e b e t t e r m e a n s t o s t u d y a c t i v i t y i n the lower portions of the digestive tract and their relation to the activities within the tureen is through the use of re-entrant cannulas. Althoug h these procedures: procedures: pres ent 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. T h e y h a v e b e e n u s e d s u c c e s s f u l l y b y Br i t i s h work ers Phillipson, 19 1952 52 ; H og an and Phillipson, 1960; Singleton, 1961; Ash, 1961a, b, 1 9 62 62 a , b ) a n d b y Do u g h e r t y 1 9 5 5 ) i n t h e Un i t e d S t a t e s . T h e s e wo r k e r s n o t o n l y d e scribed the techniques and some of their uses but also the molds for making the cannulas. The paper by Dough erty 1 9 5 5 ) p ro r o v i d es es some excellent diagrams of fistulation operations for the rumen, duodenum, abomasalduodenal shunt and caecum, as well as descriptions of the construction of cannulas.

Cannula Although several types of cannulas have been used in rumen fistulas, the

Other Fistulation Techniques Esophageal fistulation techniques and canhulas have recently been improved to the ext e n t t h a t t h is is t e c h n i q u e h a s b e c o m e h i g h l y

Rumen Volume Determination I n m a n y s t u d i e s o f a c t i v i t y wi t h in in t h e r u men, especially quantitative studies, it is deFigure 5. Two sizes of Jarrett type cannulas shown with metal collars and rubber stoppers.

South Aus Aus-5 The Ja rret t cannula is obtainable from the South trali an Rubber Mills Adela Adelaide ide South Austral ia. 6 Mould 5i1 5i1701 701 Avon India Rubber Company Ltd. Melksham Wiltshir Wiltshiree Englan d.

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s i r a b le l e t o d e t e r m i n e t h e v o l u m e o f th th e r u m e n . P e r h a p s t h e e a r li li e s t t e c h n i q u e a n d o n e w h i c h i s s t il il l u s e d o c c a s i o n a l l y i s s l a u g h t e r o f t h e a n i m a l f o ll l l o w e d b y l i g a ti ti o n o f t h e c o m p a r t ments 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. S w e d i sh sh w o r k e r s ( S p e r b e r e t a l . 1 9 5 3 ; H y d e n , 1 95 95 5 , 1 9 6 0 , 1 9 6 1 ) e m p l o y e d p o l y ethylene 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 developm e n t , t h is is t e c h n i q u e h a s b e e n w i d e l y u s e d w i t h varying degrees of success. Although the author has not used this method, users have s u g g e s te te d t h a t t h e m e t h o d d o e s n o t y i e l d r e -

most practical rations, the determination of this fraction is not easily made by indicator t e c h n i q u e s p r e s e n t l y a v a i l a b le le . S t u d i e s o n t h e distribution of chromic oxide in the rumen have been reported by Corbett and Greenh a l gh gh ( 1 9 5 9 ) a n d C o r b e t t e t a l . ( 1 9 6 0 a , b ) .

p r oRdeuccei nb tl ley ,d al ti at h iaunm d issa l vt se r hy a vt iem eb eceonn suusm e di n tgo. 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 determinat io i o n . A l th th o u g h s o m e w o r k e r s ( D . B . P u r s e r , personal communication, Ohio State University) re com men ded sampling 1 hr. after dosing sheep, workers in the author's laboratory have determined that a more representative s a m p l e is is o b t a i n e d 2 ~ t o 3 h r . a f t e r d o s in in g b e e f c a t tl tl e . W a l k e r a n d H a w l e y ( 1 9 6 5 ) c o m p a r e d l i th th i u m a n d p o l y e t h y l e n e g l y c o l i n t h e determination of rumen volume and found cl0se agreement between the two methods on t h e f i r st st d a y a f t e r d o s i n g . L i t h i u m i s a b s o r b e d , however, and partially recycled to the rumen via the saliva. The main disadvantage of the techniques d e s c r i b e d is is t h a t t h e y a r e i n d i c a t o r s p r i m a r i l y o f t h e f l u id id v o l u m e a n d n o t n e c e s s a r i l y o f t h e 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

2 . nTohner uumt ii li lni az taitnio i og n aonfi mnaalt u raanld p trhoet e i rnesl aitni o na to the nitrogen metabolism in the rumen microbial pop ulation. 3 . T h e d i f fe f e r e n t ia i a l f u n c ti ti o n s o f a n y n u m b e r of isolated bacterial species which might b e a d d e d t o t h e r u m e n i n p u r e c u l tu tu r e o r in defined mixtures. 4 . T h e d i f f e re r e n t ia i a l f u n c t io io n o f p r o t o z o a a n d bacteria. 5 . T h e r e q u i re r e m e n t s f o r th th e m a n y m i c r o n u trients and growth factors known to be required by monogastric animals.

approximately the same specific gravity and particle size as the solid ingesta. Since these t w o q u a l i ti ti e s a r e u s u a l l y h i g h l y v a r i a b l e w i t h

Gnotobiotics

T h e u s e o f g n o to t o b i o ti t i c a n im im a l s i n r u m e n s t u d i e s i s s t il il l i n i t s i n f a n c y . N e v e r t h e l e s s , t h e determination of the function and importance of the rumen microorganisms in the ruminant w i ll ll e v e n t u a l l y d e p e n d o n t h e u s e o f t h is is t y p e of technique. The number of aspects of rumin a n t n u t r i t io i o n a n d p h y s i o lo l o g y w h ic ic h c a n b e studied with this type of animal presents a c h a l le l e n g i n g a r r a y . T h e f o l lo lo w i n g r e p r e s e n t s only a partial listing: 1 . T h e r o le l e a n d n e c e s s i t y o f v o l a t il il e f a t t y acids in the ruminant.

A l t h o u g h t h e f i rs rs t g e r m - f r e e r u m i n a n t s w e r e r e p o r t e d b y K u s t e r ( 1 9 1 2 , 1 9 1 3 ) , li l i t tl tl e more was accomplished with this technique u n t il i l t h e r e p o r t b y L u c k e y ( 1 9 6 0 ) . C o n s id id e r able 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 b e g i n w i th th t h e d e l i v e r y o f n e w b o r n r u m i n a n t s 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 Univ ersity and M ichigan State Unive rsity are under the leadership of D. B . Purser and C. Smith respectively.

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In this review the author has attemp ted to d i s c u s s b o t h i n vitro a n d in vivo t e c h n i q u e s for studying rum en m icrobial activity and f u n c t io io n . F r o m t h e s t a n d p o i n t o f t h e a d v a n tages and disadv antag es of the various techniques, the po tential uses and in some cases t h e e a s e o f m a n i p u l a t i o n , a s p e c t s o f th t h e in t e c h n i q u e s b a r e b e e n d e s c r i b e d i n detail vitro t o a ll ll o w t h e r e a d e r t o a s c e r t a i n w h i c h p r o c e d u r e s m i g h t s a t i s fy h is r e q u ir e m e n t s . M o s t o f t h e in vivo t e c h n i q u e s d i s c u s s e d w e r e n o t d e s c r i b e d i n s u f f ic ic i e n t d e t a i l t o a l l o w t h e reader to use them, but h opefu lly to enable t h e r e a d e r t o s e l e ct c t th th e t e c h n i q u e s t h a t m i g h t seem most useful and subsequen tly refer to t h e l it i t e r a t u r e f o r f u r t h e r d e t a il il s . I n m o s t c a s e s the descriptions offered by the originators of the techniques would be far more accurate and helpful than those that might be given by the reviewer. I n s e l e c t io i o n o f a n in vitro t e c h n i q u e , t h e r e is no substitute for logic invo lving the obj e c t i v e o f th th e s t u d y a n d t h e i n t e r p r e t a t i o n w h i c h i s t o b e m a d e f o l l o w i n g t h e s tu tu d y . Th ese factors mu st be considered fully prior t o th e d e s i g n o f t h e e x p e r i m e n t . I n m a n y c a se s pub lished techniqu es will of necessity hav e to b e m o d i f i e d t o s a t i s f y t h e r e q u i r e m e n t s o f th th e s t u d y . T o t h e m o r e s o p h i s t ic ic a t e d b i o c h e m i s t s s o m e o f t h e t e c h n i q u e s d e s c r ib e d m a y a p p e a r c r u d e a n d l a b o r io u s w h e n c o m p a r e d w i t h t h e m o r e r e fi fi n ed 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 t o d a y . H o w e v e r , w i t h th th e c o m p l e x s y s te m that e x i s ts ts i n r u m e n fe rm entatio n these techniques m ay well serve as a prelimin ary or adju nct to mo re r e fi fi n e d s tu tu d i e s . In

value of alfalfa and timothy by varied techniques. J . D a i r y S c i . 44:2232 A s h , R . W . 1 9 6 1 a . A c i d s e c re re t io io n b y t h e a b o m a s u m and its relation to the flow of food material in sheep. J. Physiol. 156:93. A s h , R . W . 1 9 6 1 b . S t i m u l i i n f lu l u e n c i n g t h e s e c r et e t io io n o f a c i d b y t h e a b o m a s u m o f sh s h e ep ep . J . P h y s i o l . 1 5 7: 7: 185. A snul h , aRe. f W 1 9e6p.2 a J. . OPmhys a s iool - a. b 164: o m a 4. sal re-entrant canor . s he A s h, h, R . W . 1 9 6 2 b . G a s t r o - i n t e s t i n a l r e - e n t r a n t c a n nul a e f or s t udi e s of di ge s t i on i n s he e p. A n. P r od. 4: 309. Baker, F. and S. T. Harris. 1947. The role of the microflora of the alimentary tract of herbivora w i t h s pe c i a l r e f e r e nc e t o r um i na nt s . 2. M i c r obi a l d i g e s ti t i o n i n t h e r u m e n ( a n d c a e c u m ) w i t h s p e c ia ia l r e f e r e nc e t o t he de c om pos i t i on of s t r uc t ur a l c e l l ul os e . N ut r . A bs t r . R e v. 17: 3. B a k e r , T . I .,., G . u Q u i c k e , O . G . B e n t le le y , R . J o h n s o n a n d A . L . M o x o n . 1 9 5 9 . T h e i n f lu lu e n c e o f c e r t a in in phys i c a l pr ope r t i e s of pur i f i e d c e l l ul os e s a nd f or a ge c e l l ul os e s on t he i r di ge s t i bi l i t y by r um e n m i c r oo r g a n i s m s in vitro J. Animal Sci. 18:655. B a l c h, C . C . 1950. F a c t or s a f f e c t i ng t he ut i l i z a t i on of f ood by da i r y c ow s . I . T he r a t e of pa s s a ge of f ood t h r o u g h t h e d i g e s ti t i v e t r a c t . B r i ti ti s h J . N u t r . 4 : 3 6 1 . B a t c h, C . C . , a nd V . W . J ohns on. 1950. F a c t or s a f f e c t -

Adler, J. H., J. A. Dye, D. E. Boggs and H. H. Will i am am s . 1 9 5 8 . G r o w t h o f r u m e n m i c r o o r g a n i s m s i n a n in vitro c o n t i n u o u s - f l o w s y s t e m o n a p r o t e i n f r e e di e t . C o r ne l l V e t . 48: 53. Anderson, R., E. Cheng and W . Burroughs. 1956. A laboratory technique for measuring phosphorus availability of feed supplements fed to ruminants. J. Animal Sci. 15:489.

i n g t h e u t i l iz iz a t i o n o f f o o d b y d a i r y c o w s . 2 . F a c t or s i nf l ue nc i ng t he r a t e of br e a kdow n of c e l l ul os e (cotton thread) in the rumen of the cow. British J. Nu tr. 4:389. B a r n e s , R . F . 1 9 6 6 . C o l l a b o r a t i v e in vitro r u m e n f e r m e n t a t i o n s t u d i e s o n f o r a g e s u b s tr tr a t es es . J . A n i mal Sci. (Submitted for publication). Barnett, A . J. G. and R. L. Reid. 1961. Reactions in the Rumen. Ed ward Arnold, London. B a t h , D . L . , W . C . W i e r a n d D . T . T o r e l l . 1 9 5 6. 6. T h e 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. C o m p a r a t i v e a c c u r a c y o f s e v e ra ra l m e t h o d s . J . D a i r y Sci. 45 : 59. Baum gardt, B. R., M . W. Taylor and J. L. Cason. 1962b. Evaluation of forages in the laboratory. II. S i m p l i fi fi e d a r t if i f i c ia ia l r u m e n p r o c e d u r e f o r o b t a i n i n g repeatable estimates of forage nutritive value. J. D air y Sci. 45 : 62. Baumgardt, B. R. and H i Kon Oh. 1964. Evaluation of forages in the laboratory. IV. Within and among trial variability of the Wisconsin artificial rumen pr oc e dur e . J . D a i r y S c i . 47: 263. Belasco, I. J., M. F. Gribbins and D. W. Kolterman. 1958. The response of rumen microorganisms to pa s t ur e gr a s s e s a nd pr i c kl y pe a r c a c t us f ol l ow i ng f ol i a r a ppl i c a t i on of ur e a . J . A ni m a l S c i . 17: 209. B e nt l e y, O . G . 1959. A c om pa r i s on of a r t i f i c i a l r um e n techniques. Report TID-7578, Oklahoma Confere nc e , R a di oi s ot ope s i n A gr i c ul t ur e , p. 181. Bentley, O. G., R. R. Johnson, S. Vanecko and C. H. H u n t . 1 9 5 4 a . S t u d i es e s o n f a c t o r s n ee ee d e d b y r u m e n m i c r oor ga ni s m s f or c e l l ul os e di ge s t i on in vitro J An im al Sci. 13 : 581. Bentley, O. G., A. Lehmkuhl, R. R. Johnson, T. V. Hershberger and A. L. M oxon. 1954b. The cellu-

A nRn ui smo en n, .E J. .F .F a. nW d iDl e. y, s . 1Y9o5r9k. ,MNe .t a Y b o. l i s m i n t h e yL, eNwei w Archibald, J. G., H. Fenner, D. F. Owen, Jr., and H . D . B a r n e s. s . 1 9 6 1 . M e a s u r e m e n t o f th th e n u t r i t i v e

lfatty o l y t i cacids. f a c t oJ. r Am. a c t i vChem. i t y o fSoc. c e r t76:5000. a in in s h o r t - c h a i n e d B e nt l e y, O . G . , R . R . J ohns on, T . V . H e r s hbe r ge r , J . H . C l i n e a n d A . L . M o x o n . 1 9 5 5 . C e l l u l o ly ly t i c

in vitro

m s eal nv yes e s rteos pwe oc tr sk w i t h r a d ti oe ci shontioqpueess, lr ee ns tdi n tgh- ec m e l-l a n d c e l ll - f r e e s u s p e n s io io n s . I n a d d i t i o n , w e l l developed techniques for studying bacteria in pure culture exist and can be profitably u til iz i z e d in in c o n n e c t i o n w i t h s o m e o f t h e s t u d i e s outlined above. Literature

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