Elgersma, A.

Smit, H.J.

Taweel, H.Z.

English

NARCIS 

Can a natural protease inhibitor decrease protein degradation of perennial ryegrass in the rumen?

Wageningen Yield 

 Sustainable Grassland Productivity 553
Can a natural protease inhibitor decrease protein degradation of 
perennial ryegrass in the rumen? 
 
Smit H. J., Taweel H. Z. and Elgersma A. 
 
Crop and Weed Ecology Group (CWE), Department of Plant Sciences, Wageningen University, 
Haarweg 333, 6709 RZ Wageningen, The Netherlands. 
 
Abstract 
 
Recently breeders identified two populations of perennial ryegrass (Lolium perenne L.) that were 
thought to be different in protease activity. One population was bred to contain an increased 
concentration of the protease inhibitor (cystatin) and one population was bred to have a normal protease 
inhibitor concentration (control). The two populations were tested in three experiments. In the enzyme 
assay no significant difference could be found between the cystatin and the control population. It was 
surmised that the protease inhibitor was inactivated by the freeze drying process. The gas production run 
was done with fresh material. A small difference (P<0.01) between the cystatin and the control 
population was found in the final ammonia concentration in the rumen fluid. Therefore it was concluded 
that the natural protease inhibitor in the cystatin population might decrease proteolysis in ruminants. 
The in vivo experiment showed no reducing effect on the ammonia concentration in the rumen of sheep 
grazing the cystatin population. It is concluded that either populations were not different, or the effect of 
the examined protease inhibitor on proteolysis of perennial ryegrass protein in the rumen is limited.  
 
Keywords: Plant proteases, cystatin, rumen kinetics, sheep. 
 
Introduction 
 
Although large efforts have been made in the last two decades, losses of nitrogen to the environment are 
still a major problem in livestock production. The efficiency of nitrogen utilization by ruminants is low 
(16%). The rate of protein breakdown in the rumen frequently exceeds the level at which the released 
amino acids can be incorporated by the microorganisms. This is especially a problem when ruminants 
are fed on fresh grass, because grass consists of a large amount of rapidly degradable nitrogen. One 
possibility to improve nitrogen utilization by ruminants is to reduce the rate of protein degradation in the 
rumen. Very recently breeders identified, with genetic markers, the gene for cystatin (a protease 
inhibitor). Two different grass populations of perennial ryegrass (Lolium perenne L.) were created. One 
population was bred to contain an increased concentration of the protease inhibitor (cystatin) and was 
thought to slowdown the protein degradation another population was bred to have a normal protease 
inhibitor concentration (control) and was thought to have a faster protein degradation. These two 
populations were used in an enzyme assay test (exp. 1), a gas production run (exp. 2) and an in vivo 
(exp. 3) experiment to test the effect on the extent of protein degradation of perennial ryegrass in the 
rumen. 
 
Materials and Methods 
 
Experiment 1: The two populations were sown in pots and grown in the greenhouse. After two harvests, 
and 21 growing days the grass was harvested using scissors. The samples were immediately freeze 
dried, and milled through a 1mm sieve. An enzyme assay test for protein degradation as described by 
Mathis (2001) was performed. Triplicate forage samples that contained 15 mg N (≈1 g dry material) 
were weighed in 80 mL centrifuge tubes, and incubated in 60 mL of a borate-phosphate buffer solution 
(pH 8) for 1 hour at 39 ºC. Subsequently, 15 mL of separate enzyme solution (Streptomyces griseus) 
was added to the borate-phosphate buffer plus forage and incubated for 0, 4, 24 and 48 hours. Following 
the incubation, the samples were vacuum filtered through filter flocks. The residue was washed with 
 Grassland Science in Europe, Vol. 11 554
0
5
10
15
20
25
30
0 4 8 12 16 20 24
Time after incubation (hours)
N
H
3
 c
o
n
c
. 
(m
g
 l-
1
)
control
cystatin
distilled water. Flocks and residue were quantitatively brought into a Kjehldahl receiver and analyzed 
for total nitrogen. 
Experiment 2: Grass samples were taken from two established pastures, which were sown with seed 
from the two contrasting populations, at 28 days of regrowth. The grass was cut fresh at 4 cm, and cut 
into pieces of 4 cm lengths using a paper cutter. Approximately 4 g of fresh grass were weighed into 
140 ml bottles and incubated in triplicate with 81 ml of N-free medium, 5 ml of rumen fluid and 1 ml of 
reducing agent. Rumen fluid was obtained from three Holstein-Friesian bullocks offered a standard diet, 
consisting of 80% good quality hay and 20% concentrates. Samples were incubated in the solution at   
39 °C for 0, 1, 2, 3, 4, 6, 8, 10, 12 and 24 hours. Two bottles at each time point containing no substrate 
were included as a blank. Following the incubation, samples (5 mL) were taken and 5 mL of 
trichloroacitic (TCA) was added. NH3 was analyzed by the indophenol method (Searle, 1984). 
Experiment 3: Four grass fed sheep were equipped with rumen fistulas. Two grazed the cystatin and two 
grazed the control population. An experimental period lasted for 3 days. The first day was used for 
adaptation. On day 2 and 3 in the experimental period rumen liquid samples were taken during the 
photoactive period, at 7:00, 9:00, 12:00, 15:00, 18:00,  20:00 h. and 5 ml of TCA was added. After three 
days, sheep changed to the other population. In a third period the sheep changed back to the initial 
population. The rumen liquid samples were analyzed for NH3.  
Statistical analysis were done on all experiments, using the repeated measures design, with time as the 
within subjects factor and the population (cystatin or control) as between subjects factor.  
 
Results and discussion 
 
Experiment 1: During the incubation period, protein concentration was already decreased rapidly (from 
17.0 g kg-1 DM to 10.0 g kg-1 DM). Due to grinding of the samples (1mm), the soluble protein could 
easily be released. The residual protein was efficiently degradated within the first 4 hours of incubation 
in a buffer solution with an enzyme assay. After 4 hours of incubation no further degradation took place.  
No significant differences could be found between the two grass populations. It was concluded that 
natural breakdown inside the plant cells was not inhibited by the cystatin. But it might be that the 
protease inhibitor was inactivated by the drying process. Therefore, an experiment with fresh grass 
might give different results. 
Experiment 2: Figure 1 shows the NH3 concentration (mg L
-1) in the bottles in the period after 
incubation of the grass samples. The mean NH3 concentration was lower (P<0.01) in the cystatin 
population than in the control population, especially at 8, 10 and 24 hours after incubation when the 
difference was largest. This could be related to the reduced protease activity due to the protease 
inhibitor, but could also be due to the DM content and CP concentration, which were lower in the 
cystatin compared to the control population. When the curves were expressed as ammonia concentration 
per mg Ninput, no significant differences were found between the two populations. However, the NH3 
concentration at the end of the gas production run was lower (P<0.05) in the cystatin population. This 
might be related to a slightly higher WSC concentration in this population, but could also be an effect of 
the cystatin protease inhibitor. 
 
 
 
 
 
 
 
 
 
 
 
 
Figure 1. NH3 concentration (mg L
-1) of the buffer solution with rumen liquids incubated either with 
control (closed symbols) or cystatin population (open symbols). 
 
 Sustainable Grassland Productivity 555
0
20
40
60
80
100
07:00 09:00 12:00 15:00 18:00 20:00
time of the day
N
H
3
 c
o
n
c
. 
(m
g
 l
-1
)
control
cystatin
 
Experiment 3: Figure 2 shows the NH3 concentration in the rumen liquid of four sheep grazing either 
the control or the cystatin population during the day. The NH3 concentration in the rumen was at most 
time points lower (P<0.05) in the control than in the cystatin population, which was against expectation. 
It was only at the end of the day, at 20:00 h, when the NH3 concentration was increasing that the NH3 
concentration of sheep grazing the cystatin population was higher than those grazing the control; 
however this was not significant (P=0.06). The animal variation was high and more animals should be 
used in further experiments. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Figure 2. NH3 concentration (mg L
-1) of rumen liquid samples of sheep grazing either the control 
(closed symbols) or the cystatin population (open symbols). 
 
Conclusions 
 
It is concluded that either the populations were not different, or the effect of the examined protease 
inhibitor (cystatin) on proteolysis of perennial ryegrass protein in the rumen is limited. In experiments 
with protease inhibitors, fresh material should be used, because the protease inhibitor could be 
deactivated by the drying process. 
 
References 
 
Mathis C.P. (2001) A collaborative study comparing an in situ protocol with single time-point enzyme 
assays for estimating ruminal protein degradability of different forages. Animal Feed Science and 
Technology, 93, 31-42.  
Searle P.L. (1984) The berthelot or indophenol reaction and its use in the analytical-chemistry of 
nitrogen - a review. Analyst, 109, 549-568. 


Can a natural protease inhibitor decrease protein degradation of perennial ryegrass in the rumen?

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