Albanese, R.

Domenis, M.

Gugliemelli, A.

Pellikaan, W.F.

Pérez, O.N.

Smidt, H.

Tiemessen, F.

English

Name not available

Changes in methanogenic populations residing in the rumen of dairy cows in response to a sainfoin (Onobrychis viciifolia Scop.) based diet

Wageningen Yield 

198 Ruminant physiology
Changes in methanogenic populations residing in the rumen of dairy cows 
in response to a sainfoin (Onobrychis viciifolia Scop.) based diet
A. Guglielmelli1,3, O. Perez2, F. Tiemessen1, M. Domenis1, R. Albanese1,3, S. Calabrò3, H.S. Smidt2 
and W.F. Pellikaan1
1Animal Nutrition Group, Wageningen University, 6700 AH Wageningen, the Netherlands; 
2Laboratory of Microbiology, Wageningen University, 6700 AH Wageningen, the Netherlands; 
3Dipartimento di Scienze Zootecniche e Ispezione degli Alimenti, Faculty of Veterinary Medicine, 
Federico II University, 80137 Naples, Italy; Wilbert.Pellikaan@wur.nl
Introduction
Methanogenesis in the rumen is a natural metabolic process which keeps the fermentation on by 
continuously utilising the hydrogen (H2) released by fermentation of carbohydrates. Methane 
emissions by ruminants have implications for (1) animal productivity because they constitute 
a significant loss of dietary energy, and (2) the environment because methane is considered 
to be one of the more powerful greenhouses gases involved in global warming. Several plant 
secondary metabolites can be used for selective inhibition against the methanogens in the rumen. 
An antimethanogen activity has been attributed to tannins, mainly condensed tannins, present in 
sainfoin (Onobrychis viciifolia Scop.) (Waghorn et al., 2002). These polyphenolic compounds 
can reduce methane emission by reducing protozoa number and changing the rumen fermentation 
parameters. A reduction in methane production could be associated with a change in the number 
and/or in the diversity of the bacterial community in the rumen. Polyethylene glycol (PEG) has a 
strong affinity with condensed tannins present in sainfoin and it is commonly used to block their 
action. The current trial involving dairy cows was conducted to study the effect of sainfoin tannins 
on rumen fermentation and on methanogen functioning, and to assess adaptive behaviour of rumen 
microbiota when exposed to sainfoin tannins for a prolonged period.
Material and methods
Prior to receiving the experimental diet three rumen fistulated Holstein Friesian dairy cows (DIM = 
226±64 d; milk production = 29.4±6.1 kg/ d; mean ± se) were placed on a lucerne based ‘uniformity 
diet’ for a 2-wk period to allow animals to adapt to a tannin free legume-based diet. After 2 wk, 
the lucerne was exchanged for sainfoin. During the first 5 d of the sainfoin feeding polyethylene 
glycol (PEG4000; 975 g/cow/d) was administered in three portions per day through the fistula. After 
the PEG treatment the animals remained on their sainfoin based diet for 7 more weeks. Rumen 
content samples were taken three times per week during the experimental period. Samples were 
collected before the morning feeding and were prepared for analyses on fermentation end-products: 
volatile fatty acids (VFA) by gas chromatography and ammonia (NH3) by a colorimetric method. 
Ciliate protozoa were microscopically enumerated in rumen fluid samples using a Sedgewick 
Rafter counting chamber (Dehority, 1984). DNA was extracted from rumen fluid collected and 
the number of methanogenic Archea were determined in the sample using the SYBR green qPCR 
assay; Methanobrevibacter smithii was used as the reference strain. The PCR conditions and primers 
used were as reported by Denman et al. (2007). In addition, samples were taken to determine the 
diversity of the rumen methanogen population by denaturing gradient gel electrophoresis (DGGE, 
Skillman et al., 2006). Data were analysed using the GLM procedure of SAS® 9.1 (SAS Institute 
Inc., Cary, NC, USA). Differences between dietary treatments (lucerne, sainfoin+PEG, sainfoin) 
with time were tested using a repeated measures statement.
Ruminant physiology 199
Results and discussion
The results show a significant (P<0.05) decrease in the number of protozoa, and a non- significant 
decrease in the total number of Archea in the first week after changing to the sainfoin diet (Table 
1). The Archea followed a similar tendency as the protozoa but animal variation seemed to be 
considerably higher. The decrease in Archea numbers already started during the PEG administration 
period. This suggests that PEG may not have been fully successful in completely blocking the effect 
of tannins. Total VFA and NH3 followed a pattern similar to the protozoa numbers. The VFA profile 
shows some significant differences among diet treatments. The results show no differences in total 
VFA between the uniformity diet and the sainfoin + PEG diet, but a distinct and significant (P<0.05) 
drop in VFA when PEG treatment was stopped (122.5 vs. 95.67 mmol/l). In contrast, NH3 shows a 
significant (P<0.05) decrease when animals changed from uniformity diet to the sainfoin + PEG diet, 
followed by a further drop in the first few days after stopping the PEG treatment; further supporting 
our observation that PEG was not fully effective. The difference in the DGGE animals profile through 
time involving all methanogenic groups shows an inhibition in the whole methanogen community.
Table1. Dietary treatment effects on rumen fluid parameters and microbial cell count.
Treatment Protozoa (log10/ml) Number of Archea (log10/ml) Total VFA (mmol/l) NH3 (mg/l)
Lucerne 5.79a 8.14 124.5a 138.2a
Sainfoin+PEG 5.78a 7.69 122.5a 85.4bc
Sainfoin 1st wk 5.65c 7.46 95.7b 54.4b
Sainfoin 2nd wk 5.72b 7.57 114.7 109.1ac
Sainfoin 3rd wk 5.73b 7.41 105.9 94.4c
MSE 0.0006 0.45 152.1 439.5
P-value 0.0002 0.69 0.08 0.01
Means within columns followed by different superscripts are significantly different at P<0.05; 
MSE: mean square error.
Conclusion
The tannin content in Onobrychis viciifolia Scop. resulted in the inhibition of protozoa and 
number of methanogens. The tendency of the different parameters suggests that the microbial 
population (protozoa, Archea, other bacteria) responds in different ways to the sainfoin diet over 
time, suggesting that rumen microbiota adapt to the dietary conditions. Further work is required to 
evaluate the relation between the microbial inhibition and methane production.
References
Dehority, B.A., 1984. Evaluation of subsampling and fixation procedures used for counting rumen protozoa. 
Appl. Environ. Microbiol. 48: 182-185.
Denman, S.E., N.W. Tompkins and C.S. McSweeney, 2007. Quantification and diversity analysis of ruminal 
methanogenic populations in response to the antimethanogenic compound bromochloromethane. FEMS 
Microbiol. Ecol. 62: 313-322.
Skillman, L.C., P.N. Evans, C. Strompl and K.N. Joblin, 2006. 16S rDNA directed PCR primers and detection 
of methanogens in the bovine rumen. Lett. Appl. Microbiol. 42: 222-228.
Waghorn, G.C., M.H. Tavendale and D.R. Woodfield, 2002. Methanogenesis from forages fed to sheep. Proc. 
N.Z. Grassl. Assoc. 64: 167-171.


16S rDNA directed PCR primers and detection of methanogens in the bovine rumen.

Evaluation of subsampling and fixation procedures used for counting rumen protozoa.

Methanogenesis from forages fed to sheep.

Quantification and diversity analysis of ruminal methanogenic populations in response to the antimethanogenic compound bromochloromethane.

NARCIS 

Changes in methanogenic populations residing in the rumen of dairy cows in response to a sainfoin (Onobrychis viciifolia Scop.) based diet

Abstract

Similar works

Full text

Available Versions

Name not available

Wageningen Yield

NARCIS