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INTESTINAL DIGESTION OP PROTEIN - N AND UTILIZATION IN THE RUMINANT

UREA

1.2.3 INTESTINAL DIGESTION OP PROTEIN - N AND UTILIZATION IN THE RUMINANT

Interest in the digestive tract of ruminant animals has usually centred on the forestomach, for it is the characteristic digestive organ of the ruminants and about two-thirds of the dige­

stible organic matter is fermented there. The ruminant intestine has been neglected because of the assumption that it resembles that of monogastric animals in its functions. However, the oapacity of the rumen and the metabolic activities of its micro­

organisms affect the flow and composition of digesta passing to the intestines to an extent that makes intestinal digestion in ruminants a distinctive process. Kay (1

969

) showed that

(1) food is retained in the rumen for a long time and only flows to the lower gut slowly;

(2) microbial activity in the rumen transforms the diversity of protein in the diet to a more uniform product passing to the abomasum; it also removes most of the digestible carbohydrate from the food so that very little sugar is absorbed from the intestine;

(

3

) flow of digesta from abomasum is enormous, almost continuous and fairly constant in consistency and composition; pancreatic secretion is equally continuous, abomasal secretion of diges­

tive fluid is continuous and the intestinal content remains acid throughout the upper part of the small intestine.

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(

4

) large amounts of water and salts are secreted into the gut

especially b y the salivary glands and these must be efficiently re-absorbed mostly in the small and large intestines. The nitrogenous digesta flowing to the duodenum are largely of rumen microbial origin, though variously supplemented with unfermented food residues and digestive scretions. The faotors affecting the digestion of food in the intestine, therefore, are the extent of protein degradation in the rumen, the nature and quantity of microbial protein synthesized from dietary and endogenous nitrogen and the amount of endogenous c protein flowing to the duodenum.

Heclcer 0 9 7 0 compared the deaminative, ureolytic and proteo­

lytic activities and rates of cellulolysis, carbon dioxide and methane production in the rumen with that of the large intestine.

He found that the proteolytic activity of the large intestine is greater than that of the rumen contents. Some proteolytic activity was present in caecal cell-free liquor. Deaminase activity was greater in rumen than in caecal oontents. The urease activity of rumen contents was greater than that of oaeoal contents. The rate of carbon dioxide and methane production was, however, higher in oaeoal contents than in rumen contents. The rate of cellulose breakdown in vivo were similar for rumen and caecal contents.

Thus it is seen that the large intestine, though little studied, is also capable of enormous digestion, the principal difference

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4 4

being that since hydrolytic digestion of protein, starches, sugars and fats occur before the digesta reaches the caecum, the amounts of these substances reaching the caecum are likely to be small or negligible.

Hogan and Weston (1

967

) have shown that the amounts of non­

ammonia crude protein (NACP) passing the abomasum was similar whether the ration contained 7.8 or

1 9

.

8

^ crude protein (CP), ftrskov, Fraser and McDonald (1971 ) found that the amount of NACP (Y^g/day) disappearing from the small intestine increased with protein intake (X g/day) according to the equation

Y

1

= 2.12 X - 0.0057 X

2

-

83

.

reaching a maximum when there was CP in the dry matter of the feed.

Andrews and prskov (1970a) showed that when the protein concentra­

tion of the diet was increased at high constant energy intakes, the growth rate and the retention of nitrogen in the body increased The level of protein was found to have n o . significant effect on the disappearance of NACP from the large intestine. The apparent digestibility of crude protein increases with protein concentration It was not known whether increased absorption came from increase in microbial protein or from dietary nitrogen escaping fermentation Since they found that the protein used, soya be®n has higher

digestibility than microbial protein, they concluded that the

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increments were due to soy-bean protein escaping fermentation in the rumen,

1.2.3.1 The Rate of Flow of Digesta in the Digestive System of Ruminant Animals

A mathematical study of the movement of particles and solutes through the digestive tract of the ruminant has been presented b y Warner (1

966

). From his study, he showed that in a ’ steady - state' system,

F = 0.693V _ _ _ _ _ (1)

T where

F = rate of flow from the rumen.

V = volume of liquid in the rumen.

T = Time for the equivalent of half of the liquid in the rumen to be transferred to omasum.

When a water - soluble marker is infused continuously into the rumen, it was shown that

F = i / c (2)

R = v/f = 1.44 T ( 3 )

P = I X R _ _ ( 0

and X

= , (5)

where I = rate

it

of infusion of marker into the rumen.

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h6

C = concentration of marker in the liquid leaving the rumen.

R = the mean retention time of a population of marker molecules in the rumen.

P = the quantity of marker present in the rumen (the rumen marker pool).

K = is the fraction of the rumen volume transferred to omasum per unit time.

In a "steady-state system", one estimate of F, V and T may be obtained b y administering a single dose of an appropriate marker into the rumen and studying its rate of disappearance.

If the marker is infused continuously at a constant rate, a number of estimates of rate of flow from the rumen can be made from the concentration of marker in the liquid leaving the rumen b y using equation 2. After the continuous infusion is stopped, an estimate of T may be obtained b y studying the rate of dis­

appearance of marker from the rumen. The value of T together with the estimates of the rate of flow during the continuous

infusion, m a y be used to calculate the volume (v ) of water in the rumen as indicated by equation (l). It was assumed that the concentration of marker in the liquid leaving the rumen and abomasum were the same as those obtained in samples of liquor taken from those organs. Rate of flow from the abomasum was

calculated from equation (2) by substituting the marker concentration

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The digestibility coefficients of the dietary nutrient for the entire tract have frequently been obtained b y determining the ratio of a given food constituent to some indigestible marker in the food itself, such as lignin and the ratio of the cons­

tituent to the marker in the faeces. From them, the percentage of the nutrient digested is given as

100 -

f

100 X

^

T’ — ■*" --- x

^

This method does not require quantitative collection of faeces, provided representative samples can be obtained.

The markers most frequently used in ruminant digestion studies are lignin, polyethylene glycol, and chromic oxide.

Chromic oxide is used either in powdered form mixed with the ration (Drennan, Holmes and Garrett, 1970) given in gelatin capsules (Putnam, Loosli and Warner, 1956) or impregnated on to paper (Cowlishaw and Alder, 1963).

Chromic oxide has been shown to be associated with the solid phase of intestinal digesta (Harris and Phillipson, 1962) and can be easily and accurately determined. Polyethylene glycol associates itaelf with the liquid phase of digesta (Hyden, 1956)

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and the method of its determination have not given consistent results. Lignin has the advantage of being a plant constituent but suffers the disadvantage of being an ill-defined entity, the estimation of which is empirical.

Johnson, Dinuson and Bolin (

1964

) examined the concentration of chromic oxide in all the sections of the gut of sheep after feeding and measured the rate of excretion of a single dose when given in paper form or as powder mixed with a pelleted ration.

They found that the powder form moved through the gut significantly faster than the paper form and that this difference was established by different rates of passage from the rumen. Prom their results, it seemed as if the passage from omasum to abomasum of the paper form was similar to that of lignin. Consequently, chromic oxide concentration in the abomasum might be used to give an accurate estimation of digestion anterior to this point if the paper form were used. Johnson et al. (1964-) also found that the powder form yielded an abomasal concentration of chromic oxide only of that found when the paper was used and would lead to a large underestimation of digestion.

Balch (1957) used the lignin-ratio technique to determine the extent of digestion in the reticulo-rumen of the cow. The results showed that about of the herbage dry matter was digested in the reticulo-rumai, and that in cows fed entirely on hay, the amount of nitrogen flowing out of the reticulo-rumen was

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greater than the nitrogen intake. Rogertson (1958) also used the lignin-ratio technique to determine partial digestion in sections of the alimentary tract of sheep using a slaughter method. He shaved that 4Qfo> and

1 %

of the dry matter of hay, mixed diet and concentrate respectively occurred in the rumen. Bines and Davey (1970) also using the same technique found that 6($ of straw diet dry matter was digested in the rumen.

Drennan, Holmes and Barrett (1970), and Holmes, Drennan and Garrett (1970) compared the use of lignin and powdered chromic oxide as markers for estimating the magnitude of digestion in the rumen and intestines using slaughter technique in sheep. They found that the results obtained using lignin as marker was higher and more consistent than those obtained using chromic oxide

powder, and suggested that the poor results obtained b y using powdered chromic oxide might be due to its very rapid or uneven passage from the rumen. They found that about 7Q$ of the organic matter digested occurred in the rumen.

For studies with ruminants, chromic oxide paper appears to be suitable and promising, no doubt owing to the slow and sustained release of the oxide as the paper undergoes microbial digestion (Corbett, Greenhalgh, McDonald, and Florence, 1960).

This has been confirmed b y Langlands, Corbett, McDonald and Reid (

1963

) and Lambourne and Reardon (1

963

) who showed that chronic

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50

cxide in the form of impregnated paper gave a more even release of marker into the faeces.

Digestibility of nutrients in the sections of the digestive tract is also estimated by the techniques of the re-entrant

cannulation. For studies of digestion in the reticulo-rumen, the cannula is placed at the abomasum or duodenum so that all digesta from the reticulo-rumen can be collected. Digestibility of a nutrient is then calculated as the difference between the nutrient in food and the nutrient recovered at duodenal collection point.

Similarly digestion in the small intestine is determined by

placing cannulae at duodenum and terminal ileum, and the nutrient passing through the duodenal cqnnula nanus the nutrient reaching the terminal ileum is the amount of nutrient apparently digested in the small intestine. Digestion in large intestine is the difference between the total nutrient in terminal ileal point and in the faeces. Digesta may be totally collected at the collection points or samples of digesta may be collected at suitable intervals and pooled to give representative samples of digesta flowing through the portion of digestive tract.

Chromic oxide either in powdered form or impregnated on to paper, or lignin is usually given so that digesta could be adjusted to percent recovery of the marker. Tills technique of the re-entrant

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cannulation has been widely used (Topps, K a y and G-oodal, 1968;

Nicholson and Sutton, 1969; McRae and Armstrong 1970; and McRae, J970). It is not only useful for determining digestibility in

sections of the digestive tract but also in studying biochemical reactions in the sections of the digestive tract, thus Hecker (1971) used sheep with ruminal and caecal cannulae to compare metabolism in the rumen and the caecum.

Using the re-entrant cannulation method for determining digestibility, Hogan and Phillipson (i

960

) found that of the total dry matter digested in the sheep,

7

C$ disappeared in the stomach,

11

$ in the small intestine and

19

$ in the large intes­

tine whereas the corresponding values as obtained b y Topps

et

a l . (

1968

) are

6

l$,

22

$ and

11

$ for hay, and

65

$,

11

$ and

11

$ for

ooncentrate - fed animals in the stomach, small intestine and large intestine respectively. Several investigators (Nicholson and Sutton, 1969; Topps at .al.,

1968

) have reported that more nitrogen is recovered at abomasum than fed when sheep are given diets low in nitrogen but that substantial loss of nitrogen occurs in the rumen when the diet is rich in nitrogen.

Ben - Ghedalia, Tagari and Bondi (1974), b y means of cannulae placed in portions of the small intestine, were able to show that there were substantial increases in water, dry

matter and total nitrogen in the section immediately distal to the

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52

pylorus and that these were caused b y the inflow of bile, and pancreatic and duodenal juices. The net increase found beyond the entry of the common bile duct was 2.7g protein N and 2.0g non-protein N per day. The region 7 - 1 5m from the pylorus was found to be the region of most intensive absorption of amino acids,

60.5

of the essential, and l*.j$ of the non-essential amino acids passing through the region being absorbed. They also shovred that only small changes occurred in the region after

15

m distance from the pylorus.

McRae, Ulyatt, Pearce and Hendtlass (1972), in ten 21*. hr.

collections of digesta entering the duodenum and eleven 21*. hr.

collections of digesta reaching the ileum of sheep given dried grass showed that there were highly significant correlations between the 21*. hr. flows of chromium marker and the corresponding

flows of dry matter, organic matter, nitrogen, gross energy, hemicellulose and cellulose at both sites. This has enabled the investigators to estimate the quantitative intestinal digestion in sheep.

The reactions in the digestive tract are very complex and a knowledge of how they take place, the products formed, the utili­

zation of the products formed, and the factors that enhance the production is essential for adequate feeding of ruminant animals and hence meat production.

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ISOTOPIC METHODS OF DETE R M I N I N G THE U T I L I Z A T I O N OF N I T R O G E N IN THE R U M INANT

The isotopes that have been c o m monly used in nutri t i o n

15 35

studies are n either in urea or a m m o n i u m salt form, S, u sually used as sulphate, and 32P us u a l l y in the form of phosphates- These isotopes have been used to study the synthesis or util i z a t i o n of p rotein in the ruminant. In

I if

addition C is also used to study the util i z a t i o n of c a r ­ bohydrates or other carbon- b e a r i n g materials. It is used

1

b

either as C in urea or in glucose. White, Steel, Len g and 1

b

L u i k (

1969

) have u sed C glucose to study the kinetics of glucose m e t a b o l i s m in the sheep. Harrison, Beever and Thomson (1972) and Beever, H a r r i s o n and Thomson (1972) have used 35S as sodium sulphate to estimate the p r o p o r t i o n of food and microbial p rotein in the duodenum of the sheep, while Landis

(

1968

) h a d also use d s ulphur -

35

as so d i u m sulphate to s t u d y quantitative aspects of sulphur m e t a b o l i s m in the ruminant.

M a t h i s o n and M i l l i g a n (1971) and Nolan and L e n g (1972) have used 15N as A m m o n i u m chloride or sulphate to study the ruminant

digestion, while L a n d and V i r t a n e n (1959) have used 15 as A m m o n i u m n i trate to st u d y the synthesis of milk p r otein from A m m o n i u m salts. L o f g r e e n and K l eiber (1953) used 32P or N: P 32 ratio to determine the value of the metab o l i c faecal n i t rogen of you n g calves

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All the isotopes in common use satis f i e d the basic r e q u i r e ­ ment that:

(a) the c o m pound s t udied for instance urea, ammonia, glucose can be label l e d in the required p o s ition with a suitable isotope,

(b) the label is firmly attached to the molecule or or at least to that part of the molecule which is of interest to the investigator,

(c) the amount of isotopic material introduced into the initial compound is such that it allows for considerable dilution before the c o n c e n t r a ­ tion of the isotope is too low for accurate determination,

(d) when radioactive, the rate of decay is suf f i c i e n t l y low to permit all the r a d i o a c t i v i t y determinations to be made with r e asonable accuracy, while on

the other hand, the r a d i o a c t i v i t y does ncrfe- persist s u f f i c i e n t l y l e n g and with s ufficient

intensity to cause significant radia t i o n damage to the tissues or cells under i n vestigation or to any part of the experimental animal during the course of the experiment, otherwise the experiment is not truly physiological.

5k

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The isotopic tracer method is one suited par e x c e l ­ lence for the study of b i o c hemical reactions in the living cells. The major advantages of the method are:

(a) The experiment can often be carried out under s t r i c t l y p h y s i o logical conditions on intact normal 'A

a n i m a l s .

(b) With a few exceptions, the l a b elled compound has, for all purposes, the same biological properties and the same metabolic fate as the unlab e l l e d compound.

(c) The amount of isotope required is u sually e x t r e ­ mely small, p a r t i c u l a r l y with r a d ioactive isotopes.

(d) Laborious s e p a r a t i o n of r a d i oactive compounds from tissues and tissue extracts is often unnecessary.

(e) The precise origin of individual atoms in a compound p r o duced by living tissues can oft*n be deter m i n e d by isotope studies for instance the .N cf urea in blood or urine, the S or P atoms in proteins.

There are some limitations, however, even though these are not of such a character as to reduce s e r i o u s l y the value of the isotopic tracer method as a general technique for a g r i c ultural research. These d isadvantages are:

(a) There is n eed for s pecial technique and s p e c i a l i z e d equipment and these are u sually expensive, for example Geiger counter, Mass spectrometer, E m i s ­ sion spectrometer. The compounds themselves for

”15 32

instance Cl or Na^ SO^ are expensive.

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56 (b)

(c)

The method only involves follo w i n g the label. The d etermination gives a measure of the amount of label in the sample analysed. It should not be a u t o m a ­ t ically concluded that the amount of isotope present in a p articular tissue or cell gives a true measure of the con c e n t r a t i o n in that tissue of the substance o riginally a d m i n i s t e r e d to the animal. Also the isotope determinations give no direct information about the fate of any n o n - l a b e l l e d parts of labelled molecules which have undergone disruption. These difficulties can often be overcome by the use of two or three different labels attached to different parts of the molecule of the compound studied, for example, N i t r o g e n and S u lphur in Ammon i u m

-52S0^4

R a d ioactive isotopes may cause serious r a d i ation damage to the tissues. This may apply to the tissues of the experimental animal or of the e x p e r i m e n t e r .

« In the former case, the experiment may no longer be normal or physiological, and the results obtained may be largely due to a d i sordered m e t a b o l i s m of the irradiated or some i ndirectly affected tissues.

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(d ) L a c k of a suitable i s o t o p e

-There are few instances where it is impossible to find a suitable isotopic tracer for a biological investigation- Sometimes, however, a radioactive isotope which might otherwise be suitable has

rate of decay of a c t ivity whi c h is too short or too lon g for the experiment planned. In the former case, there would, be great difficulty in comp l e t i n g all the r a d ioactive m e a s u rements before the l a b elled compound and its m e t abolites lose their r a d i o a c t i v i t y and in the latter case there will be a c o r r e s p o n d ­ ingly greater risk of r a d i a t i o n damage to the tissues.

(e) Chemical n o n - i d e n t i t y of isotopes may not be strictly true. The p h y s i c o - c h e m i c a l differences between

the isotope used as a label and the most abundant stable isotope of the same element may o c c a s i o n a ­ lly be s u f f i c i e n t l y great to cause significant, quantitative differences b e tween the metab o l i s m of the labelled and u n l a b e l l e d compound, for example, He a v y water D^O pene t r a t e s into red blood cells more slowly than does o r d i n a r y water.

However, there is no evidence that these 'isotope effects' are n o r mally of great magnitude in the complex biochemical

systems of animal and plant organisms.

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5 8

Isotopic methods have been exte n s i v e l y used in the study of u t i l i z a t i o n of ruminal a m monia and blood urea by rumen micro-organisms. M a t h i s o n and M i l l i g a n (1971) used the isotopic tracer technique to determine the p r oportion of microbial p r otein derived from ruminal ammonia. 15NH^CI solution (2L/2^ hour) was c o n t i nuously i nfused for periods of

120

-

216

hours into the rumen of sheep which were allowed to feed 2 out of every 10 minutes. These treatments achieved

'steady metabolic states' in the r u m e n in the per i o d of the investigation. They found that 50 - 65% of bacterial N and 31 - 5 5 % of protozoal N were derived from ruminal ammoniaj

60

- 92% of the daily N intake was tran s f o r m e d into ammonia, and 17 -

5^% of

the a mmonia formed was absorbed. The g e n e r a ­ tion time of bacterial p r otein was found to be be t w e e n

38

and

hours. The investigators showed that increase in ruminal a m monia leads to a decrease in the conversion of p rotein into a m m o n i a in the rumen , a n d was given b y this relationship:

Y = 123 -

O.kk

x where

Y = N i t r o g e n c o n v e r t e d into a mmonia expressed as perce n t a g e of N intake.

X = C o n c e n t r a t i o n of ruminal a m monia (mg NH^ - N/litre).

These results were similar to those of Pilgrim, Gra y and Weller (1970). Nolan and L e n g (1972) used isotopic dilution

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