ANKOM Daisy - Manual (Page 14)

Appendix D (continued)
14
Preparation of Inoculum and Incubation:
Maintain all glassware at 39°C
(a) Preheat two 2L thermos bottles by filling with 39° C water. Empty heated water just prior to
collection of rumen inoculum. Using the appropriate collection procedure, remove at least
2000 ml of rumen inoculum and place in thermos. Include approximately two "fistfuls" of
the fibrous mat from the rumen with your collection in one thermos.
(b) Preheat a blender by filling with 39P o
P C water. Empty the heated water just prior to pouring
the rumen inoculum from the thermos into the blender. Purge the blender container with COB
2 B
gas and blend at a high speed for 30 seconds. The blending action serves to dislodge mi-
crobes that are attached to the mat and assure a representative microbial population for the in
vitro fermentation. Filter the blended digesta through four layers of cheesecloth into a five-
liter flask (pre-heated 39° C). Filter the remaining rumen fluid in the other thermos through
four fresh layers of cheesecloth into the same five-liter flask. NOTE: Allow for extra cheese-
cloth around the edges to facilitate squeezing contents of filtered mat. The flask should be
continually purged with COB
2 B
and continued during the transfer of the inoculum.
(c) Remove one digestion jar from the DaisyP II
P Incubator and add the 400ml of inoculum to the
buffer solution and samples. Purge the digestion jar with COB
2 B
gas for thirty seconds and se-
cure lid.
(d) Repeat process for all digestion jars to be used. NOTE: Do not allow COB
2 B
gas to bubble
through the buffered inoculum, rather use the COB
2 B
to form a gaseous blanket over the con-
tents of the jar.
(e) Incubate for 48 hours. The DAISYP II
P Incubator will maintain a temperature of 39.5°C ± 0.5.
If temperature of jars varies greater than one degree then move incubator to a warmer loca-
tion or place blanket or similar insulator over incubator.
(f) At completion of incubation, remove jars and drain fluid. Rinse bags thoroughly with cold
tap water until water is clear. Use a minimum of mechanical agitation.
(g) When determining True Digestibility it is necessary to remove microbial debris and any re-
maining soluble fractions using Neutral Detergent Solution. After rinsing the bags in water
place them in the ANKOMP
200
P Fiber Analyzer and follow the procedure for determining
NDF. Record the post in vitro NDF weight as WB
3 B
. NOTE: Bags can be stored in the refrig-
erator or freezer until NDF determinations can be performed.
D. Calculate:
% IVTD (as received basis) = 100 – (W
B
3
B
- (W
B
1
B
x C
B
1
B
)) x 100
W
B
2
B
% IVTD
B
DM
B
(DM basis) = 100 – (W
B
3
B
-
B B
(W
B
1
B
x C
B
1
B
)) x 100
(W
B
2
B
x DM)
Where: WB
1 B
= Bag tare weight
WB
2 B
= Sample weight
WB
3 B
= Final bag weight after In Vitro and sequential ND treatment
CB
1 B
= Blank bag correction (final oven-dried weight/original blank bag weight)