MATERI ALS AND METHODS

I nt ro du c t i on

I n v i t ro fermentation t e chniques for ob t ainin g org�ni c mat t e r

dige s t i b i l i ty w e r e carri ed o u t u s i ng the m e thod o f Til l ey and Terry ( 1 963 ) , as modi f i e d by Al exander and McGowan ( 1 96 1 ) whe re c entri fuging

was replac e d by fil tration and organic mat t e r was use d inst ead of D . M . for d i g e s t ib i l i ty calculat ions .

6 . 1 . General

6 . 1 . 1 .

Ab out 1 k g . ( D . M . b asis ) o f l e a fy spring pasture grass

( p e r e nnial ry e grass dominant ) was c u t with hand c lippers , dri e d in a forc e d draught oven at 65°C . for 48 hours and ground to pass a 2 . 0 mm . s i e v e using a W i l ey mil l . Thi s was then div i d e d into two par t s a f t e r thorough mixing and one part was a gain ground to pass a 1 . 0 mm . s i ev e . The s e samples w e re s t ored at - 1 0 C . 0 until r equired . Th e l a t t e r s amp l e s were use d throughout th e expe riment unle s s otherwis e

s t a t e d .

6 . 1 . 2 . out

S amples ( 0 . 5 gm . ) o f the s tandard grass were w e i gh e d into 1 00 m l . , glass b o t t l es ( di ame t er 5 c m . , h e i gh t 1 0 c m . , and diame t e r o f n e c k 4 c m . ) with 6 to 1 0 repl i c a t e s a c c ording t o requirements . A t the same t im e tripli c at e samp l e s w e r e w e i gh e d for mois ture and organic mat t e r

C o l l e c tion of rumen

The c o l le c t ion o f rume n l i quor from the c ows was s t andardis e d

as far as possib l e , i . e . , t ime , type o f f e e d and posi t i o n o f rum e n . Rumen li quor w a s st rained through f o u r lay e rs o f muslin i n t o a plastic

0

b o t t l e kept at 39 C . immedia t e ly b e fore it was requi r e d and further

fil tered through a p i e c e o f mus lin into a b e aker i n the l ab oratory . pH was measured and rec orded within 1 0 min . a f t e r th e c o l l e c t i on o f rumen liquor .

6 . 1 . 4 . First fermentation

Forty ml . of arti f i c ial saliva ( McDouga l , 1 949 ) , gass ed with

C02 and the pH adjusted to 6 . 90 to 7 . 00 by using normal sodium

c arb onat e solution ( N - Na2co3 ) , was added to all bot t l e s inc luding b lanks , which contained only rumen liquor and art i fi c ial sal iva . The

b o t t l e s and c on t e n t s were warmed in a water bath b e fo r e the rumen

inoculum was added. Ten ml . o f rumen l i quor was adde d t o a l l b o t t l e s which w e r e gassed w i th co2 f o r ab out 2 0 s e c onds and c l o s e d w i th a rub b e r bung fi t t e d with a gas r e l e ase valv e made on th e princ iple o f

t h e Bunse n v alv e . A l l b o t t l e s we re incubat e d i n a t h e rmostati cal ly

controll e d wat e r bath at 38 . 5°C . + 0 . 5 c . 0 for 48 hours .

During the fermentation period , the b ot t l e s w e re occasionally swi r l e d ( 5 - 6 t im e s ) and pH was m e asured a f t e r 6 , 1 2 , 24 and 48 hour s , and i f n e c essary adjus t e d t o pH 6 . 9 with N - Na2co3 s o l u t i o n . The b o t t l e s w e re again flush e d out w i th C02 b e fore ret urning them t o the wat e r b a t h . I t was found , howev e r , that adjusting the pH was n o t nec e s sary w i th this· e xperiment and this p r o c e dure w a s n o t u s e d any

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6 . 1 . 5 . S e c ond fermentation

At the end of first stage f e rmentat ion , pH was l ow e r e d t o 1 . 2

- 1 . 3 w i th 20% ( v/v ) H C l , and 5 ml . o f aqueous p epsin was added t o each

b o t t l e (0.03 gm . of 1 : 2 , 500 pepsin ) .

All b o t t l e s w e r e again flush e d wi th co2 , mix e d thorough ly , cap p e d and returned to the wat e r bath for a further 48 hours . Anaero- bic c onditions were not n e c e ssary at this stage , howev e r , sat urat ion with co2 l e s s e n e d frothing. Thi s s e c ond st age digesti on was d e s i gne d t o c omp l e t e protein degradation .

6 . 1 . 6 . F i l t ra t i on and final

At the end of 48 hours f e rmentation with pepsi n , app roxima t e ly

1 gm. o f fi l t e r-aid (Hy flo sup e r c e ll ) was added to a l l b o t t l e s and

the c ontents o f the bot t l e s were fi l t ered on to glass fibr e f i l t e r

pap e r , using H o r t l ey 3-p i e c e funnels. T h e residue w a s wash e d four t im e s with h o t wat e r .

The fi l t e r pap ers with r e si du e s w e r e dri e d a t 1 00°C . for 1 6

h ours and we i gh e d (Wa) . Th ey were then i gni t e d at 550°C. for 4 hours

i n a mu f f l e furnac e , and reweigh e d ( Wb ) . The form e r r ep r e s e n t s dry w e i ght o f th e f i l t e r pap e r , fi l t er-aid and grass sampl e undi g e s t e d during t h e i n v itro fermentation a n d the lat t e r rep resents the w e i gh t o f f i l t e r pape r , f i l t e r-aid and inorganic mat t e r o f grass s ampl e undige s t ed , sinc e n o w e i gh t changes were obtained b e fore and a f t e r i gn i tion o f fil t e r pap e r and f i l t e r- ai d .

The p erc entage dige s t i b i l i ty o f the feed organic mat t e r i n v i tro was c al cu l a t e d by sub trac t i n g the residual o r ganic mat t e r ( Wa Wb ) , l e s s the b lank organic mat t e r from the feed o rganic m at t er and

::_� . 4 1 - ' ..

. .. ""

expre ssing this as a p e rc entage o f feed organ i c mat t e r as foll ows : % di g e s tibility o f O . M . i n in v i tro =

O . M . in ( ( Wa-Wb ) for - m e an o f ( Wa -Wb ) for b lank ) O . M . in 0 . 5000 g sampl e

6 . 2 . t rial

6 . 2 . 1 . Inoculum taken from a c ow on sev eral

For thi s experim ent a lac tating c ow with a rumen fistulae ( c ow N o . 48 ) was grazed on pasture alone wi th th e r e s t of the h erd . Rumen l i quor was c o l l e c t e d at 9 : 00 a . m . from the c ow whi ch had b e en k e p t i n the yards s i n c e the morning milking ( 6 : 00 a . m . ) . Eigh t to t en repli- cates of s t an dard grass sampl es and two to four repl i c a t e s of b lanks , a c c ording to the wat e r bath c apac i ty , w e r e incubat e d a c c ording to the

m e thod prev i ously desc ribe d . Four runs were made on four separa t e days

and resul t s were analys e d to d e t e rmine the repe atab i l i ty o f the me thod and day -t o - day variation .

6 . 2 . 2 . I n o c ulum t aken from a c ow fed on s ev e ral

S i n c e no c on t ro l on intake , time o f f e e ding , wat e r intake and

uni formi ty of diet were possib l e w i th grazing animals , a fistulated dry c ow ( No . 1 20 ) was fed wi th medium qua l i ty hay ( hay B , s e e Part I ) . The c ow was f e d hay for 1 0 days b e fore c o l l ec t i on o f rumen l i qu o r . Thi s c ow was taken from t h e yard whe r e hay was f e d a d lib . to she d at 6 : 00

a . m . and rumen liquor was coll e c t ed a t 9 : 00 a . m .

S i x t o t e n repl i c a t e s o f s tandard grass w i t h four b lanks w e r e incubate d i n t h e wat e r bath for 5 s eparat e runs o n di f fe r e n t day s . The r e su l t s were analys e d t o d e t e rmine the repeatab ili ty of the m e th o d and

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day -to-day v ariati on .

o f sourc e o f i noculum - inoc ulum ' vs

inocul um�

To e liminate any day - t o - day v ariation , t wo sourc e s o f inocula w e r e c ompare d w i thin an e xperim e n t . C o w 48 and c o w 1 20 we re av ailab l e f o r this s t udy . Cow 48 was gra z e d on pasture and cow 1 20 was f e d the hay descri b e d previously . The two sou r c e s of i no culum we re c ompare d in t erms o f in v i t ro organic mat t e r d i g e s t i b i l i ty o f the s t andard grass.

Th e design o f this e xperiment was as fol l ows :

6.4.

S ourc e s o f rumen li quor ( 2 ) x standard gras s sampl e ( 6 )

+ 2 x 4 b l anks = 20 b o t t l e s .

o f two c ows as donors of rumen

Two f i s t u l a t e d lac t a t i n g c ows were av ailab l e for thi s study (c ows No . 4 8 , 4 5 ) . Both c ows w e r e grazed on t h e sam e area as other mi lking cows t o ensure that simi lar qual i ty of past ure was e at e n. Rumen l iquor was c o l l e c t e d by fis t u l ae at 9 : 00 a . m . from b o th c ows whi ch had b e e n l e f t in the shed s i n c e th e morning m i lki ng. The rumen liquors w e re c ompare d by m e asuring fermentation losses using the s t andard gra s s as sub s t rat e . Th e d esign o f this experiment was as follow s :

S ourc e s o f rume n l i quor ( 2 c ows) x s t andard grass

sample ( 1 0 repli c a t e s) + 2 c ows x 4 b lanks = 28 b o t tl e s . Time o f rumen c o l l e c ti on i n relation to time sinc e last

A fistul a t e d cow ( N o . 48 ) was availab l e for thi s study . The c ow was b rought into the she d at 8 : 00 a . m . and the rumen li quor was c o l l e c t e d at 1 0 : 00 a . m . , 2 : 00 p . m . and 8 : 00 p . m . , i . e . , 2 , 6 and 1 2 hours a f t e r grazing. Four repl i c a t e s of the s t andard grass samp l e w i th t ripl i c a t e blanks w e r e inc luded in each t r eatment , i . e . ,

6 . 6 .

6 . 6 . 1 .

three treatments ( 3 ) x sub stra t e ( 4 r eplicat e s ) + three t reatm e n t s ( 3 ) x 3 b lanks = 2 1 b ot t l e s .

I n vi tro s t udi e s w i t h B and C

o f for in v i t ro

When each hay was chopp e d and mixed on the c onc r e t e floor for th e in v ivo di ge s t i b i l i ty trial ( se e Part I ) , a b ulk samp l e was taken

from 80 d i f ferent points a f t e r thorough mixing. The s e b ulk samp l e s

from e ach hay were thoroughly mixe d agai n separat ely a n d finally a sub ­ samp l e o f approxima t e ly 1 k g . was taken from the 1 0 k g . b ulk samp l e ( he r e a f t e r cal l e d hay sample s ). The h ay sampl e s were dri e d a t 65 °C . for 1 6 hours and ground using the sam e m e thod for prepara t i o n as the s tandard grass samp l e ( se e 6 . 1 . 1 . ) .

6 . 6 . 2. Sourc e o f inoc ulum

The fistulat e d cow ( N o . 48 ) was used throughout this experi­ ment as rumen l iquor donor animal . Thi s cow was gra z e d o n pasture . The pre liminary study ( 6 . 4 . ) showed that there was no s i gn i f i c an t di f ference on i n v i t ro digestibility o f the standard gra s s whe n grass inoculum was c ompar e d w i th hay inoculum and u sing grass i n o c u l um was more c onv e n i e n t i n p rac t i c e than u s i ng hay inoculum sinc e no indoor

7 4

f e e ding w a s requi re d . Rumen l iquor was c o l l e c te d 2 hours aft e r the c ow was b rough t into the she d , as no si gni ficant di f ferenc e was obtained in v i t ro dig e s t i b i lity o f the sandard grass in re lation t o t ime sinc e

grazing ( 6 . 5 . ) .

A l though pasture was r e s t ri c t e d b e c ause o f w i n t e r shortage ,

no hay supp l ement was fed.

o f second f e rme ntat ion

Thi s experiment was d e s i gned to see whe th e r the s e c ond s tage of fermentat ion c ould b e short e ne d .

The expe rimental d e s i gn was a s follows : a ) F e rmen ta t i on p eriods c ompare d :

4 8 hrs ( 1 s t stage ) + 48 hrs ( 2nd st age ) ( p epsin )

4 8 hrs 1 1 + 2 4 hrs 1 1

b ) F e e d samp l e s use d :

Hay A , B , C , and the standard grass . c ) R e p li c at i o n :

Four repli cates for each o f the hay s

Thr e e replicate s f o r th e standard grass and blanks.

6 . 6 . 4 . o f fi rst fermentation

The aim o f this e xp e riment was t o d e t e rm i n e the e f fe c t o f l e ngth o f fermentat ion o n o rgani c mat t e r digestib i l i ty i n v i t ro and i t s relation to o rganic matt e r dige s t i b i l i ty i n v iv o .