‫بسم هللا الرحمن الرحيم‬

Comparison of protein fermentation characteristics in

rumen fluid determined with the gas production
technique and the nylon bag technique

By : M.A.Al-badwi
Prof. Dr. Ahmed M. El-Waziry
Introduction

Protein is one of the most important Components of

rommant diets

Protein metabolism in the rumen is the result of

metabolic activity of ruminal microorganisms.

Ruminants have protein requirement at two levels:-

- firstly, to meet the nitrogen (N) needs of rumen
microbes.
- secondly, to meet the amino acids requirements of
the host animal .
Evaluation of Nutritive Value of Ruminant Feeds
1. Chemical analysis:

Proximate Analysis Van Soest Fiber

Air-dry Feed Sample Air-dry Feed Sample

Boil with neutral

detergent, pH = 7
Dry Matter Crude Protein
o
• dry at 105 C • Kjeldahl N
• N x 6.25 Neutral Detergent Fiber Neutral Detergent Solubles
cellulose, hemicellulose, cell contents and pectin
and lignin
Ash
• combust sample at
o
500 C Crude Fiber
Boil in acid
• Boil in weak acid,
detergent, pH = 0
then boil in weak
base
• fiber is insoluble
fraction
Ether Extract
• boil sample in ether Acid Detergent Fiber Acid Detergent Solubles
• fat is soluble cellulose and lignin hemicellulose

Rinse in 72% sulfuric acid

Lignin Cellulose
(dissolved)
1.1. Near infrared reflectance spectroscopy (NIRS).

1.2. Electrophoretic analysis .

2. In vivo methods

2.1- Digestion trials .

2.2- In Situ technique(nylon bag technique or in sacco

technique)
In Situ technique
The principle of in situ method .
Standard technique in many feed evaluation systems .
Determine rate and extent of feed degradation in the rumen.
Divides a feed sample in a washout (W), (U) and (D) fraction.
The advantage :
- That degradation characteristics of all chemical components in
a feed can be investigated.
Disadvantages:
- very laborious and so expensive
a) Sample size to bag surface ratio
b) Bag pore size
c) Diet of the host animal
d) Number and species of animals
f )Sample preparation
g) Washing the bags
h) Interpretation of results
3. In vitro methods:
Techniques that measure feed composition, digestibility of
nutrients, feed degradation, ruminal fermentation and passage
rate from the rumen as following:

3.1- The two stages technique (Tilley & Terry, 1963).

3.2- The rumen simulating technique (Rusitec), 1977.
3.3- Enzymatic method .
3.4- In vitro gas production technique .
In vitro gas production technique
Anaerobic digestion of carbohydrates by ruminal microbes
produces VFA, C02, CH4 ,and traces of H2 . So, measurement of
gas production in vitro can be used to study the rate and extent
of digestion of feedstuffs.
Developed to estimate protein fermentation in the rumen.
The advantage :
It is quick and cheap, and many data can be collected in one run.

a) Sample preparation and size.

b) Buffer .
c) Incubation time & time of reading.
 In situ measurements
Three mature rumen
fistulated cows

fed ad libitum a ration containing:

720 g/kg forage (DM basis), including on a
DM basis
Grass hay (23 g/kg)
Grass silage (143 g/kg)
Maize silage (437 g/kg)
Pea silage (65 g/kg)
Maize gluten meal (127 g/kg)
Corncob silage (96 g/kg) feeds were incubated at the
Solvent extracted soybean meal (52 g/kg) same time (3, 8, 16, 48 and 336 h) bag/cow 35
Solvent extracted rapeseed meal (52 g/kg) Sample 5 g
Minerals (5 g/kg)

collected , washed for (40 min) ,dried at 70 ◦C (24 h(

analysed for DM, ash and CP

The washout fraction (W) of N (CP) was determined by washing with cold tap water. The residue of N after 336 h incubation
was considered to be the undegradable fraction (U). The degradable fraction (D) was calculated as 1 − W − U.
 In situ measurements
Two non-lactating rumen fistulated

fed on a diet composed of 1 kg of

standard compound feed
containing :
- 150 g/kg starch, offered in the
morning, and ad libitum
-grass hay in the morning and
afternoon.
-Animals had free a ccess to
water and to mineral–vitamin Rumen fluid samples (after the morning feeding )
blocks

Rumen fluid was strained through two layers of cheesecloth

Pre-warmed insulated flasks, previously filled with CO2, and kept at 39◦ C under CO2
 buffer/mineral solution
- N-free and contained.
- To avoid a too high input of N from the rumen fluid.

Rumen fluid was diluted 1:19 with the buffer/mineral

solution.
Added to the buffered rumen fluid rapidly fermentable
carbohydrates and incubated at 39 ◦C to be certain that N
was the limiting factor to fermentation.
 pre-incubation was performed in
- IN bottle 5 l with continuous flushing of CO2 .
- In four small bottles in the gas production equipment, with 60 ml
buffered rumen fluid to follow the gas production.

After only 4 h of incubation, gas production had ceased.

All laboratory handlings were carried out under continuous

flushing with CO2 .

All samples were incubated in one run in duplicate.

Gas production was recorded for 72 h, using a fully automated
system
Results:1. Feed samples
2. In situ degradation of CP
3. Gas production incubations :
Discussion:

That the excess of rapidly fermentable

carbohydrates depleted all obtainable N.
Carbohydrates never became limiting.
Fermentation after the maximum capacity
of the buffer did not occur.
N was limiting and stopped fermentation.
The gas production for casein and urea
(Fig. 3)longer period than incubations with
the other samples (Fig. 4a and b).
The N in the feed samples was partly
soluble (Fig. 1), which means that the
other part is not soluble.
The gas production for casein showed
higher gas productions at higher amounts
of casein per bottle, was not seen for urea.
Casein and N in the feed samples contain
amino acids, whereas urea is a non-
protein N source.
All urea samples (15–100 mg N), gas
production stopped completely after 15–20
h.
This implies that the microorganisms need
to synthesize these amino acids
themselves, needing energy for this
process.
Most of the gas production appeared to indicate
a linear gas production between 5 and 20 h of
incubation.
Gas production at different incubation periods
correlated with the results obtained by the nylon
bag incubations (Table 2).
Highest correlations between the W-fraction and
gas production were obtained after 5 h of
incubation (r2 = 0.63), and between the rate
of degradation (kd) and gas production after 20–
30 h of incubation (r2= 0.49–0.51).
There was high correlation between the
calculated amount of rumen escape protein
(REP, Table 2) and the gas production after
12-25 h (r2= 0.83-0.85).
The relationship between the amount of REP
and the gas production after 20 h (r2= 0.85) is
shown in Fig. 5
Fig. 5. Relationship between the amount of rumen escape protein (g/kg CP), as determined with
the in situ nylon bag technique,and the gas production per 15mgN after 20 h of incubation ( r2 =
0.85).
Conclusions :
The gas production technique was originally
developed to determine fermentation kinetics
of organic matter in rumen fluid.
The gas production technique can be adapted
to study protein fermentation kinetics in rumen
fluid, by using a N free buffer and using an
excess of rapidly fermentable carbohydrates.
There was a close relationship (r2= 0.85)
between the gas production and the amount of
rumen escape protein, determined with the
nylon bag technique
‫السالم عليكم ورحمة هللا وبركاته‬