Grain and artificial stimulation of the rumen change the abundance and diversity of methanogens ...

140 Pages·2008·0.92 MB·English

Grain and artificial stimulation of the rumen change
the abundance and diversity of methanogens and their
association with ciliates

by
Claus Thagaard Christophersen
Candidatus Agronomiae (M.Sc.)

This thesis is presented for the degree of Doctor of Philosophy
of The University of Western Australia

Animal Science
School of Animal Biology
Faculty of Natural and Agricultural Sciences
December, 2007
“We create the future by citing the past”
(Unknown)

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Declaration
Declaration
The work presented in this thesis is my own work except where stated below. This work
was carried out in the School of Animal Biology at the University of Western Australia
and at CSIRO Livestock Industries at Floreat Park. The material presented in this thesis
has not been presented for any other degree.
The volatile fatty acids analysed were by the Department of Agriculture, WA.

Claus Thagaard Christophersen, December, 2007.
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Publications
Publications arising from this thesis
Refereed publications:
- Christophersen C.T., Wright, A-D.G. and Vercoe, P.E. (2007) Methane emission and
acetate/propionate ratio decreased when artificial stimulation of the rumen wall are
combined with increasing grain diets. J. Anim. Sci. in press
Chapter 4 in this thesis is identical to the paper published in Journal of Animal Science
except that the abstract has been removed.

- Christophersen, C. T., Wright, A-D.G. and Vercoe, P. E. (2004). Examining
diversity of free-living methanogens and those associated with protozoa in the rumen. J.
Anim. Feed Sci. 13: 51 – 54.

Conference abstracts
- Christophersen, C. T., Wright, A-D. G. and Vercoe, P. E. (2004). Does dietary
manipulation change the diversity of methanogens and protozoa that interact within the
rumen? 4th joint INRA-RRI Symposium, Gut Microbiology – Concerns and Responses
to food safety, Health and Environmental issues, 21-23 June 2004, Clermont-Ferrand,
France. J. Repro. Nutri. Develop. 44: 52.

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Acknowledgements
Acknowledgements
Here at the very end of my PhD I would like to thank several people, who have helped
me through and been there when times were tough. Without the financial support from
The University of Western Australia (IPRS) and the Danish Research Agency, it would
never have been possible for me to undertake my PhD candidature. I am very grateful
for being granted such a possibility. I was also awarded a University of Western
Australia travel grant to attend the INRA-RRI 2004 gut microbes conference. For this I
am very grateful.
Huge thanks to my supervisors, Dr. Philip E. Vercoe (UWA) and Dr. Andre-Denis G.
Wright, for not just being supervisors but also becoming great friends. They have both
strongly supported my scientific research intellectually, and they are excellent
supervisors and have wonderful personalities. Their encouragement and understanding
have been invaluable.
I also would like to thank Mr. John Beesley for his great support and guidance. John
was always happy to share his 30+ years of experience working with animals, and he
would also be the first to put his hand up when samples had to be collected. The
measurements of liquid and particulate matters retention time had not been possible
without him.
Dr. Clare Engelke, thank you for being such a good friend and for taking samples and
looking after the animals while I was away, you are a true “life saver”.
My unofficial co-supervisors Dr. Lucy C. Skillman and Dr. Richard Cookson I also owe
great thanks for taking the time to listen, discuss and answer all my questions.
Numerous people have provided help, advice, encouragement and friendship during my
PhD and it is impossible to mention all of them. However, I would like in particular to
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Acknowledgements
thank: Mr. Andrew Toovey, Mr. Peter Hutton, Dr. Ian Williams, Dr. Suzanne Rea, Mrs.
Carolyn Pimm, Mr. Andrew Williams, Dr. Zoey Durmic and Mrs. Margaret Blackberry.
Thanks to my parents, Birthe and Ove Thagaard Nielsen, for supporting me in achieving
my goals and for visiting us here “down-under”. I would also like to thank my parents-
in-law, especially my mother-in-law for visiting us in Perth so many times, and for all
the washing and cleaning and baby sitting, making life with two PhDs and two boys
much easier for us.
The love, encouragement and support from my lovely wife, Helle Martha
Christophersen, have been outstanding. Words just cannot express how grateful I am for
that.

Claus Thagaard Christophersen, December, 2007.
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Abstract
Abstract
In Australia, there is pressure to reduce the amount of methane produced by ruminant
livestock because they are the single largest source of methane emitted from
anthropogenic sources, accounting for 70.7% of agricultural methane emissions. In
addition, methane production represents a loss of gross energy intake to the animal. The
organisms that are responsible for methane production in the animal gut are a distinct
group of Archaea called methanogens. Methanogens occupy three different niches
within the rumen. Some live freely in the rumen digesta (planktonic), others are
attached to the outer surface of the rumen ciliates (ectosymbiotic), and some reside
within the ciliates (endosymbiotic). The types and number of methanogens, as well as
rumen ciliates and their symbiotic interactions, influence the amount of methane
produced from the rumen. These factors in turn are affected by many factors, including
diet and ruminal retention time. In this thesis, I tested the general hypothesis that
increasing the amount of grain in the diet and reducing the retention time would affect
the abundance and diversity of methanogens in their different niches, including their
association with ruminal ciliates.
Twenty-four fistulated sheep were used in a complete factorial design with the
sheep randomly divided into four groups. The sheep had a 2-wk acclimatization period
on an oaten-chaff diet, followed by three, 3-wk diet phases. In diet phase 1 all sheep
were given the same oaten-chaff diet. Two of the four groups were maintained on the
oaten-chaff diet for the duration of the experiment with pot scrubbers added to the
rumen of one of the two groups. The remaining two groups were offered a low grain
diet (35% grain) in the second diet phase followed by a high grain diet (70% grain) in
the third diet phase. Pot scrubbers were also added to the rumen of one of these two
groups of grain-fed sheep. The pot scrubbers were inserted with the intention of
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Abstract
increasing rumen stimulation without changing the diet composition. Ruminal pH,
volatile fatty acids was measured to monitor rumen fermentation. In addition, methane
production (in vitro) and ruminal retention time (in vivo) were measured in the 4
treatment groups at the end of each diet phase. A robust real-time PCR assay was
developed to quantify methanogens and a denaturing gradient gel electrophoresis
(DGGE) assay was developed to examine the diversity of methanogens. Rumen ciliates
were also examined using the same methods but with already published protocols. The
DGGE gels were analysed using Gelcompar II and the resulting DGGE banding
patterns were analysed using a multivariate statistical software program (PRIMER). The
Shannon index for each treatment group was also calculated based on the banding
patterns to indicate the direction of diversity changes.
The addition of grain and pot scrubbers changed rumen parameters. Methane
production in vitro was reduced on a low grain diet in sheep with pot scrubbers.
Methane production was reduced in sheep fed the high grain diet, with or without pot
scrubbers. Acetate/propionate ratios were also lower in sheep fed the high grain diet.
The total abundance of methanogens and ciliates in diet phase 3 was not different
between treatments. However, the abundance of methanogens associated
endosymbiotically with rumen ciliates was significantly higher in high grain-fed sheep
with and without pot scrubbers.
Fifteen of the most dominant methanogen DGGE bands were sequenced and
identified using a BLAST search. Ten of the 15 different bands had >98% identity to
Methanobrevibacter spp., whereas the other five bands were found to be between 82%
and 96% similar to Methanobrevibacter spp. The diversity of methanogens and ciliates
were also found to vary between treatments. The change in DGGE banding patterns and
Shannon indices when sheep were fed grain indicated that the types of methanogens
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Abstract
changed when sheep were fed low and high grain diets, but their diversity did not. In
contrast, the diversity of rumen ciliates decreased when sheep were fed a high grain
diet. A total of 18 bands from the DGGE analysis of the ciliates were sequenced. All
except one, which was 98% similar to Cycloposthium sp. not found previously in the
rumen, matched the sequences for previously identified rumen ciliates. Some of the
rumen ciliates identified were not present in sheep fed the high grain diet.
On a high grain diet, methanogens associate endosymbiotically with rumen
ciliates to get better access to hydrogen. It appears that the association between
methanogens and rumen ciliates is dictated by the availability of hydrogen in the rumen
and not the generic composition of the ciliate population. Furthermore, endosymbiotic
methanogens appear to produce less methane than methanogens in other niches. The pot
scrubbers did not change ruminal retention time but they did reduce the
acetate/propionate measurements observed in sheep on the high grain treatment. The
reason why pot scrubbers had this effect remains unknown, but it is interesting to
consider that some physical interaction has occurred between the pot scrubbers, the
grain and the sheep that has improved the fermentation parameters in sheep fed a high
grain diet. The results from this study have advanced our understanding of the
interaction between methanogens and ruminal ciliates, and methanogenesis in the rumen
in response to dietary changes and mechanical challenges. Extending this work to look
more specifically at the species of methanogens that are most closely linked to high
methane production and how they interact with the ruminal ciliates will be critical for
manipulating enteric greenhouse gas emissions.
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Table of content
Table of content
Chapter 1: General introduction..............................................................................................................1
Chapter 2: Literature review....................................................................................................................4
2.1 Introduction.......................................................................................................................................5
2.2 Taxonomy and characteristics of rumen methanogens......................................................................6
Taxonomy......................................................................................................................................7
Pathways of methanogenesis.........................................................................................................8
Substrate range...............................................................................................................................9
Ecology........................................................................................................................................11
2.3 Taxonomy and characteristics of rumen ciliates..............................................................................12
2.3.1 The entodiniomorphid ciliates.................................................................................................13
Digestion and metabolism of dietary components.......................................................................13
2.3.2 The vestibuliferid ciliates........................................................................................................14
Digestion and metabolism of dietary components.......................................................................14
2.3.3 Interrelationships between species of rumen ciliates...............................................................15
2.4 Interaction between methanogens and rumen ciliates.....................................................................17
Niches occupied by methanogens in the rumen...........................................................................17
Why is there an interaction between ciliates and methanogens?.................................................17
Methanogens and ciliate species that associate............................................................................19
Time after feeding and the interaction between ciliates and methanogens..................................20
2.5 Grain diets and ruminal retention times influence hydrogen availability, methanogens, rumen
ciliates and their association..................................................................................................................22
Hydrogen availability...................................................................................................................22
Methanogens, rumen ciliates and their interaction.......................................................................23
2.6 Value and limitations of two molecular methods for examining methanogens and ciliates in the
rumen.....................................................................................................................................................27
2.6.1 Denaturing gradient gel electrophoresis (DGGE)....................................................................27
2.6.2 Quantitative real-time PCR (real-time PCR)...........................................................................32
2.7 Summary.........................................................................................................................................36
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