mihaela giuburunc in vitro effects of some plant … · tested to reduce the ruminal acidose and...
TRANSCRIPT
UNIVERSITY OF AGRICULTURAL SCIENCES AND VETERINARYMEDICINE CLUJ-NAPOCA
UASVM PhD SCHOOLFACULTY OF ANIMAL SCIENCE AND BIOTECHNOLOGIES
MIHAELA GIUBURUNCĂ
IN VITRO EFFECTS OF SOME PLANT SECONDARYMETABOLITES AND OF IgY IMMUNOGLOBULINS ON
GASES EMISSIONS AT RUMINANTS
SUMMARY OF THE PhD THESIS
SCIENTIFIC COORDINATOR
Prof. MIREŞAN VIOARA Ph.D.
CLUJ-NAPOCA2015
ii
TABLE OF CONTENT
INTRODUCTION................................................................................................................................... IV
PART IBIBLIOGRAPHIC STUDY
CHAPTER I ............................................................................................................................................. V
EMISSIONS OF RUMINAL METHANE- A GLOBAL PROBLEM .................................................... V
CHAPTER II ...........................................................................................................................................VI
ANATOMICAL AND PHISIOLOGICAL CHARACTERIZATION OF THE RUMEN ANDMECHANISM OF RUMINAL GAS PRODUCTION...........................................................................VI
CHAPTER III.........................................................................................................................................VII
RUMINAL METHANOGENESIS PROCESS .....................................................................................VII
CHAPTER IV ...................................................................................................................................... VIII
POLYPHENOLS AND IGY IMMUNOGLOBULINS USED IN RUMINAL METHANE EMISSIONSREDUCTION....................................................................................................................................... VIII
CHAPTER V........................................................................................................................................... IX
STRATEGIES TO REDUCE RUMINAL METHANE EMISSIONS ................................................... IX
PART IIORIGINAL RESEARCH
CHAPETR VI .......................................................................................................................................... X
THE ORGANIZATION OF EXPERIMENTS AND RESEARCH DEVELOPMENT.......................... X
CHAPTER VII ..................................................................................................................................... XIII
THE EXPERIMENTAL INCUBATION PROTOCOL OF RUMINAL CULTURES ....................... XIII
CHAPTER VIII.................................................................................................................................... XIII
TESTING THE SECONDARY PLANT METABOLITES ON RUMINAL FERMENTATIONPARAMETERS ADDED IN SINGLE DOSE IN VITRO ................................................................... XIII
CHAPTER IX ....................................................................................................................................... XV
TESTING THE PLANT SECONDARY METABOLITES ON THE FERMENTATIONPARAMETERS IN RUMINAL CULTURES ADDED PERIODICALLY IN VITRO ....................... XV
CHAPTER X........................................................................................................................................ XVI
TESTINFG OF IGY SPECIFIC IMMUNOGLOBULINS ON THE FERMENTATIONPARAMETERS IN RUMINAL CULTURES IN VITRO.................................................................... XVI
iii
CHAPTER XI ................................................................................................................................... XVIII
MOLECULAR CHARACTERIZATION OF RUMINAL METHANOGENS COMMUNITY ..... XVIII
CHAPTER XII ..................................................................................................................................... XXI
GENERAL CONCLUSIONS ..............................................................................................................XXI
SELECTIVE BIBLIOGRAPHY....................................................................................................... XXIII
iv
SUMMARYof the PhD thesis
IN VITRO EFFECTS OF SOME PLANT PLANT SECONDARY METABOLITES AND OF IgYIMMUNOGLOBULINS ON GASES EMISSIONS AT RUMINANTS
developed by Mihaela Giuburuncă, under the scientific coordination of Prof. Vioara Mireșan Ph.D.,
from University Of Agricultural Sciences And Veterinary Medicine, Cluj-Napoca
The PhD Thesis “In vitro effects of some plant plant secondary metabolites and of IgY
immunoglobulin on gases emissions at ruminants” consists of two parts, divided into twelve chapters.
INTRODUCTION
The activity of animal husbandry intensified in the last decade due to the economic boom,
urbanization and modernization of farms. The large number of ruminants is based in big part on the fact
that they have the unique capacity to convert the organic compounds in products that are consumed by
people on a daily basis.
Because of the fermentation happening in the rumen, certain gases are formed and then
eliminated by the animal into the atmosphere. The ruminal gases are correlated with the activity of the
microorganisms existing in the rumen; the production of methane gas then signifiges a great loss of the
energy intake by the animal.
The emissions of ruminal methane are a global problem, and thus in the last decade strategies
for reducing the quantity of emission of such gases have been developed. Most of the time, the
strategies are limited to adjusting the feed to the phisiology of the animal, and also to the management
of the farming practices. The research in this field started with the first in vitro tests, and in vivo
testings demostrated that certain methods can be applied on a daily basis and for a longer period of time
succesfully.
Our research is contributing to the development of these strategies and sheds light on this issue
at the national level, as the field of animal husbandry is growing within our borders.
The most well-researched strategies of reducing the ruminal methane are those that entail the
usage of feeding additives and replaicing the animal nutrition, and also the ones that utilize the
v
biotechnologies such as the creation of a vaccine against methanogens or the elimination of ruminal
protozoa.
Utilizing plant extracts to hamper the methanogens activity has become a well-reasearched
strategy that has yielded great results. The plant extracts have animicrobian and antioxidant properties
already utilized in traditional medicine. Involving these at the farm level can mean lower costs than
utilizing other strategies.
Another approach in regards to developing a strategy of reducing the ruminal methane
emissions consists in utilizing the aviary IgY antibodies. Thus in ruminants, the IgY antibodies were
tested to reduce the ruminal acidose and the effects of ruminal methanogenesis process.
Research in vitro and in vivo shows promising results, though the transformation of the ruminal
ecosistem to reduce methane emissions is a scientific challenge that needs to take into consideration the
effects it has on the health and well beeing of the animal, and also on the farm management, in regards
to profits.
PART IBIBLIOGRAPHIC STUDY
CHAPTER IEMISSIONS OF RUMINAL METHANE- A GLOBAL PROBLEM
This chapter explores the impact on climate change by the animal husbandry. According to
FAO, agriculture is responsible of approximately 14% of greenhouse gas emissions. A significant part
of these emissions are methane gas; their contribution is 23 times stronger than carbon dioxide and they
comprise 16 % of the total of greenhouse emissions.
Ruminants have the unique capacity to convert the cellulose from plant into meat, milk, wool
and leather, without entering into competition with human nutrition. Methane gas is formed in one of
the four abdomenal compartiments (in rumen ) of the ruminants due to certain microorganisms called
„methanogens”, which form a group in the Archaea domain. Owing to the production of this methane
gas in the rumen, the ruminants lose up to 15 % of their energy, thus making their digestive process
less than 100% efficient.
vi
A research made to guage the global rate of methane emissions produced by the cattles raised in
Romania during 1938-1989, concluded that during a 24 hour period, 184 liters of methane gas were
produced on average by cows, and 162 liters by calves (Zorzoliu and Zorzoliu, 1992).
According to EPA, the anual methane gas emissions produced by enteric fermentation have
risen to 4.3 % during 1990 -2007, though fluctuations were also observed during this period (1994 and
2004 showed lower values). This effect can be attributed to the development of the agricultural sector,
specifically the one involving raising meat cattle (www.epa.gov). In Europe, as well as in Romania,
emphasis was put on the developement of this sector of animal husbandry in order to insure the
production of meat and milk, and also to improve their quality.
A major factor that influences the emissions of ruminal methane is specifically the quality of
the animal’s nutrition, such as the carbohidrates types and the shift of the ruminal microflora (Lascano
and Cárdenas, 2010). The food consumed by the animals is fermented by bacteria, protozoa,
methanogens, fungus; the polysaccharides from forages are converted into volatile fatty acids and
microbian protein, accompanied by the emission of gassy products (Lascano and Cárdenas, 2010).
Sejian et al., 2011, studied and classified certain factors that influence the production of ruminal
methane gas, such as: species, reproduction, composition, proportion and forage source, quantity of
digestible nutrients, feeding strategies, growth stress, management practicies, acetate to propionate
ratio, ruminal microorganisms, and the ruminal pH (Lascano and Cárdenas, 2010).
CHAPTER IIANATOMICAL AND PHISIOLOGICAL CHARACTERIZATION OF THE RUMEN AND
MECHANISM OF RUMINAL GAS PRODUCTION
This chapter highlights the anatomical and physiological characteristics of the ruminants, in
order to better understand the processes that take place in the rumen. Unlike the monogastric animals,
ruminants contain three pre-gastric compartments: rumen, reticulum and omasum. Of these, the most
important and well developed is the rumen, whose microfauna and microflora permits the feeding to be
mostly fiberous.
The rumen is a large organ that occupies alomost the entire half of the left abdominal cavity in
ruminants (Mireșan et al., 2003). Many times it has been descriebed as a „fermentation vat” (Moran,
2005). Due to the presence of the digestive tube, ruminants have the posibility to swallow fast and to
vii
store a large quantity of forages, digestion following at a slower pace (Mireșan et al., 2003). Ruminal
microorganisms use components from the forage for their own requirements, but once they are digested
in the stomach and the intestine, they provied the hosting animal with the microbian protein. They
decompose the forage into fermeted products, such as hydrogen, acetate, propionat, butyrate. A part of
these products are absorbed by the ruminal epithelium, and are thus used as energy by the animal
(Janssen, 2010).
Large quantities of VFA are produced in the rumen, especialy acetic , propionic and butyric
acids (Bergman et al., 1965), and they are mostly absorbed in the ruminal mucose (Mireşan and
Mireşan, 1997).
The rumen contains a large variety of bacterial species that were classified and named over time
by many scientists, such as Marvin Bryant who in 1959 published his reasearch on the species of the
ruminal microorganisms. They are microorganisms facultative anaerobic (Aerobacter aerogenes,
Escherichia coli, S. liquefaciens), anaerobic microorgansims (Lactobacillus lactis, C. sporegenes),
methanogenic microorganisms (Methanobrevibacter ruminantium, M. smithii) and protozoa.
CHAPTER IIIRUMINAL METHANOGENESIS PROCESS
This chapter emphasis ruminal methanogenesis process, which is important in achievieng a
strategy in mitigation of methane emissions. Generally, methanogensis is the biological process of
methane production by methanogens. These microorganisms produce the larger part of methane gas,
which is estimated to 5 x 1014 g CH4/year (www.ncbi.nlm.nih.gov). Other microorgansims which
produce methane gas are some Eubacteria spp., though only methanogens have been reported to couple
methane generation to energy production (Sirohi și colab., 2010).
Methanogenesis process is conducted with the help of unique enzymes, such as: Coenzyme 420
– the (N-(N-L-lactyl-y-glutamyl) L-glutamic acid phosphodiester of 7, 8-didemethyl) 8-hydroxy-5-
deazaribofl avin 5 phosphate, F420 which is a low potential electron carrier and it is similar to
nicotinamide cofactors, Coenzyme M - the CoM (2-mercaptoethanesulfonic acid), which is the smallest
known organic cofactor and till 1999 was considered unique to methanogens, Coenzyme B – it is a
colorless cofactor. It was earlier called component B as it was identified as one of the three
chromatographically separated fractions required to reconstitute MCR (Methyl Coenzyme reductase).
viii
Methanofuran (MFR or carbon dioxide reduction factor) is the only cofactor known to contain furan
moiety and Methanopterin which, structurally, is related to folic acid and, in the process of
methanogenesis, acts as intermediate C1 carrier in the reduction of formyl group to methyl group.
CHAPTER IVPOLYPHENOLS AND IgY IMMUNOGLOBULINS USED IN RUMINAL METHANE
EMISSIONS REDUCTION
This chapter presents some characteristics of plant extracts used for the reduction of ruminal
CH4 emissions, and the characteristics of avian IgY immunoglobulins which are increasingly studied in
ruminal gas emission reduction. Plant extracts, along with their secondary metabolites, have been used
for centuries in traditional medicine and industry; most often they are used as food preservatives.
Phenolic acids are polyphenols compounds which can be distinguished in two classes: benzoic acid and
cinnamic acid. Hydroxybenzoic acids are components of complex structures such as hydrolysable
tannins (gallotannins in mangoes and ellagitannins in red fruits) (Hasna El Gharrras, 2009). Jayanegara
et. al. (2010) made some studies which tested the effects of several sources of polyphenols, including
simple phenols such as: benzoic acid, cinnamic acid, caffeic acid, p-coumaric acid, phenylacetic acid in
two different concentrations (2 and 5 mM) against rumen methanogenesis. They found that no
compound used in the lowest concentration (2 mM) had an effect on the production of gas, whereas
higher concentration (5 mM) had significant effects.
Tannins act on ruminal methanogens in two ways: they have a direct effect on the
microorganisms but also an indirect effect on the production of hydrogen. Further tests with tannins
extracted from Lotus corniculatus, Lotus penduculatus and Acacia mearnsii used against ruminal
methanogens have shown a reduction in the amount of methane produced by about 30% without
altering the digestibility of small ruminants (sheep, alpaca, goat) (Pinares-Patino et al., 2003c; Carulla
et al., 2005; Puchala et al., 2005). More recently it was reported that the inclusion of fortified vegetable
tannins in the diet of lambs reduces the amount of methane produced by 24%, but this is associated
with reduction of organic matter (Tiemann et al., 2008).
In ruminants, avian IgY antibodies were tested to mitigate the risk of ruminal acidosis and to
reduce the effects of ruminal methanogenesis. The results of the tests where antibodies were used
against ruminal methanogens (Methanobrevibacter smithii, Methanobrevibacter ruminantium and
Methanosphaera stadtmanae) showed that they decreased the amount of methane in vitro (p ≤ 0.05) but
ix
the effect was fleeting and, following 24 hours, the results were similar to those of the control samples
(Marcq et al.,2010).
CHAPTER VSTRATEGIES TO REDUCE RUMINAL METHANE EMISSIONS
This chapter highlights some strategies to reduce ruminal methane emission and also wants to
present some examples of studies conducted both in vitro and in vivo. The ideal goal of such strategies
is the reduction of methane gas production (liters / day) per animal. However, considering the current
system of livestock, the immediate goal should be to reduce methane per unit of product (milk or
meat).
Some strategies that use the biotechnologies are exploited, including the realization of a vaccine
against three species of microorganisms in rumen methanogens which reduced the amount of methane
by almost 8% (Wright et al., 2004). Passive immunization with avian antibodies (IgY) against three
rumen methanogens has recently been evaluated in vitro. For example, a treatment that uses the entire
egg has significantly decreased methane production in vitro, but the effect was lost after 24 hours of
incubation (Cook et al., 2008).
Using probiotics as methane gas mitigation strategy is an interesting approach. Reductive
acetogenesis serves as a natural mechanism wherein the hydrogen coexists with methanogenesis in the
gastrointestinal tract of many animals. In the rumen, acetogens are less numerous and less efficient than
the methanogens. Acetogenic used as probiotics was tested, but the results were not satisfactory or
inconclusive (Martin et al., 2009).
In ruminal ecosystem, the methanogens live in association with protozoa; thus, this system
favors the transfer of hydrogen (Martin et al., 2009). It was estimated that methanogens who are
attached to protozoa, are contributing between 9 and 37% in ruminal methanogenesis (Martin et al.,
2009). Protozoa defaunation seems very interesting but this option should be evaluated in terms of
performance for livestock. The absence of protozoa in the rumen can have different effects on the
animals which may be either positive or negative, depending on the diet and this type of production.
Until now there have been no practical techniques of defaunation (Martin et al., 2009).
x
The use of feed additives has been extensively researched and applied in countries such as New
Zealand or Australia for several years, having positive effects on productivity and the environment.
Among feed additives, antibiotics ionophores, such as Monensin or Lasalocid were used to improve
feed efficiency in livestock, and are known to diminish ruminal methane production (Martin et al.,
2009).
From the beginning, animal nutrition was a very important topic in all studies. It was concluded
that all types of feed have an impact on ruminal methanogenesis process and the use of certain types of
feed can have significant effects on it.
PART IIORIGINAL RESEARCH
The second part of this PhD thesis highlights scientific conducted over the years of doctoral
studies. It is organized in 7 chapters, all experiments made by modern research methods. Additionally,
it lays out the results, conclusions and discussions from these experiments.
CHAPETR VITHE ORGANIZATION OF EXPERIMENTS AND RESEARCH DEVELOPMENT
This chapter highlights the aim and the objectives of the PhD thesis, the original elements of the
thesis, the organization of experimental device, and also the conducted experiments.
6.1 THE AIM AND THE GENERAL OBJECTIVES OF THE EXPERIMENTS
The aim of this thesis was to identify the effects of some plant extracts and of avian IgY
antibodies on ruminal fermentation process in vitro. The plant extracts used in these experiments are
plant secondary metabolites and are represented by the following phenolic acids: trans-cinnamic acid,
cafeic acid, p-coumaric acid and the flavonoid catechin hydrate.
The objectives of the thesis are:
1. experiments organized in the laboratory in order to achieve a ruminal microcosmos;
2. the acquisition of the biological material necessary for incubation;
3. culture substrate preparation by grinding;
xi
4. culture media preparation, which is necessary in incubation of rumen methanogens;
5. realization of the stock solutions used in experiments;
6. choice of plant secondary metabolites and realization of desired concentrations;
7. the preparation of an antigen needed to produce the avian IgY antibodies;
8. immunization of laying hens with the antigen;
9. isolation of IgY antibodies from egg;
10. realization of the ruminal microcosmos;
11. study, in the first instance, the effect of four plant metabolites added to the ruminal
culture, as a single dose at a concentration of 6 mM;
12. measuring the total volume of gas produced after 24, 48 and 72 hours of incubation;
13. determining the composition of the gas with gas chromatographic method after 24, 48
and 72 hours of incubation;
14. determining the concentration of volatile fatty acids by HPLC method after 24, 48 and
72 hours of incubation;
15. determining the pH after 24, 48 and 72 hours of incubation;
16. study, in the second phase, the effects of two plant secondary metabolites added to
ruminal culture in a single dose in double concentration;
17. determining the parameters mentioned above after 24, 48 and 72 hours of incubation;
18. study the effects of two plant secondary metabolites periodically added to ruminal
cultures, by determining the parameters proposed;
19. IgY immunoglobulins testing by adding them as a single dose, and periodically at
ruminal cultures;
20. determining the parameters of interest (referred to above) after 24, 48 and 72 hours of
incubation;
21. isolation and quantification of genomic DNA from the samples taken from the culture
bottles;
22. amplification of mcrA gene by PCR;
23. molecular-biological characterization of ruminal methanogens using T-RFLP
technique;
24. statistical analysis and interpretation of results.
xii
6.2 THE ORIGINAL ELEMENTS OF THE PhD THESIS
In this thesis, we consider the elements of originality to be the following:
the choice of experimental model and organizational procedures;
the choice of plant secondary metabolites and their concentrations, of avian IgY
antibodies, as well as analyses for a prolonged period up to 72 hours;
determining some of the most important parameters of the rumen (the volume of gas
produced, its composition, pH and volatile fatty acid concentrations);
use of plant secondary metabolites and antibodies in single dose or periodically to
determine more accurately the effects of these;
identifying the Archaea community of microorganisms by molecular- biology
techniques.
6.3 ORGANIZATION OF THE EXPERIMENTS
All experiments, except the ones concerning the production and isolation of the specific IgY
immunoglobulins, were created in the department of Environmental Microbiology and Bioenergy labs
in the center of Environmental reasearch “Helmholtz Zentrum für Umweltforschung UFZ” in
cooperation with the center for biomass “Deutsches Biomasseforschungszentrum DBFZ” from Leipzig,
Germany. They span over 2013-2014.
In order to reach the objetives proposed for this PhD thesis, we established a ruminal anaerobic
microcosmos by utilizing certain special techniques. Thus, for the preparation of the ruminal
microcosmos, we used an anoxic chamber and a laminar flow hood, accesing the Hungate technique.
The biological material used was fresh ruminal liquid collected through the method of ruminal
fistula from sheep feed with dried alfalfa hay. The sheep belong to the Clinic of Veterinary Medicine
from leipzig University in Germany. The ruminal liquid was collected at the begining of every
experiment before administering the morning feedings in thermic bottles, and transported directly to the
lab.
As substrate for the ruminal cultures we used dried alfalfa hay cut in fine particles (10 mm).
The hay provided the main nutrition to the sheep from which the fresh ruminal liquid was collected.
xiii
Culture media for methanogens is supposed to offer them the necessary nutrients and to
establish the phisical and chemical conditions necessary to raise the pH level, anaerobical condition
and redox level. Thus, the culture media used was Media for Methanogens 141 DSM (Deutsche
Sammlung für Mikroorganismen und Zellkulturen GmbH).
The plant extracts that were tested in the experiments are the plant secondary metabolites, the
phenolic acids: cafeic acid, trans-cinnamic acid, p-coumaric acid and the flavonoid catechin hydrate,
procurred from Sigma Aldrich Chemie GmbH, Germany. Together with these extracts we studied the
effect of aviary IgY immunoglobulins, these were isolated in the Immunology lab from UASVM Cluj-
Napoca.
CHAPTER VIITHE EXPERIMENTAL INCUBATION PROTOCOL OF RUMINAL CULTURES
This chapter refers to the characterization of the experimental protocol of incubation for
ruminal cultures in vitro. This protocol used is described as follows:
collecting the ruminal liquid before the morning feedings from fistulated sheep;
straining the ruminal liquid through two sets of sterile cheese cloth and storing it in anoxic
conditions;
measuring the ruminal liquid in 100 ml seric bottles which contain the necessary substrata;
adding the culture media to the bottles;
adding plant extracts and IgY antibodies to the bottles;
incubating the culture bottles at 39̊ C;
at point 0, in the experiment, then again at 24, 48, 72 h, establishing the parameters for the
volume of the gas produced after point 0, the composition of the gas with GC, the concentration
of VFA with HPLC, and the pH level.
CHAPTER VIIITESTING THE SECONDARY PLANT METABOLITES ON RUMINAL FERMENTATION
PARAMETERS ADDED IN SINGLE DOSE IN VITRO
8.1 THE AIM AND THE OBJECTIVES OF THE EXPERIMENT
The aim of these experiments was to test four plant extracts represented by the secondary plant
mebolites (trans-cinnmic acid, caffeic acid, p-coumaric acid and catechin hydrate) on gas
xiv
production and on ruminal fermentation parameters in vitro. They were initially tested in 6 mM
concentration, and as a result of this initiall testing, two of them were eliminated (the trans-
cinnamic acid and catechin hydrate). We then experimented with double concentration of the
compounds. The trans-cinnamic acid and catechin hydrate extracts were eliminated because they
showed significant growth of methane gas.
The initial results were analyzed in Excel with the help of GraphPad Prism 6 programm (one
way ANOVA), and the differncies between the media were analized with the Duncan test.
8.2 RESULTS AND DISCUSSIONS
8.2.1 The effects of plant secondary metabolites added in 6 mM concentration
The experiment to which we added to the ruminal culture in a concentration of 6 mM the
caffeic acid and p-coumaric acid, showed that these two acids had a tendancy to decrease the
quantity of methane gas (Giuburuncă et al., 2014) (after 24 hours, the control sample showed an
average value of 5.51 ml/100 ml CH4, the value beeing higher than the other samples, more exact:
4,08 ml/100 ml for caffeic acid, 4.07 ml/100 ml for p-coumaric acid, 5.47 ml/100 ml for catechin
hydrate and 5.51 ml/100 ml for the trans-cinnamic acid), but these differences were not deemed
significant. After 48 and 72 hours we did not observe siginificant differences. The profile of
volatile fatty acids (VFA) studied in this experiment did not suffer significant changes (Giuburuncă
et al., 2014). We observed lower average levels for the samples in which we added the phenolic
acids, though these were not statistically significant. The pH level for all the samples was not
affected by these phenolic acids. For all the other gases emited in the ruminal culture, such as
hydrogen and carbon dioxid, we did not note any significant effects. The results of these
experiment led us to eliminate two secondary metabolites, the trans-cinnamic acid and catechin
hydrate, and to continue the experiments only with cafeic acid and p-coumaric acid, as they had the
best results concerning the production of methane gas.
8.2.2 The effects of secondary plant metabolites added in 12 mM concentration
After analizing the results of the first experiment, we decided to start a new experiment in
which the concentration should be doubled. We then decided to leave out the trans-cinnamic acid
and catechin hydrate.
xv
The secondary plant metabolites (caffeic and p-coumaric acids) added in single dose to the
ruminal cultures in 12 mM concentration showed significant decrease of CH4 emissions after 24 h
of incubation. The control had an average value of 4.91 ml/100ml CH4; the caffeic acid showed an
average value of 3.40 ml/100 ml; and the p-coumaric acid showed an average value of 3.48 ml/100
ml CH4. Emissions after 48 hours of incubation showed a decrease value only for the caffeic acid.
The average value for the control sample was 8.07 ml/100 ml CH4, while the average value for the
caffeic acid was 6.48 ml/100 ml CH4, the differinces beeing statistically significant. The values
after 72 h of incubation confirmed the fact that the effect if these two extracts is not significant on
the ruminal methane emissions. The quantity of hydrogen and carbon dioxide was not affected by
these two extracts. Regarding the VFA profile, we observed significant differences on the
concentration of iso-butyrate, the p-coumaric acid raising its value (average value for the control
sample was 2.41 mg/l, and the concentration for the p-coumaric acid was 3.96 mg/l after 24 h).
After 48 hours of incubation, we observed significant results: adding the p-coumaric acid resulted
in a lower concentration of acetate and the caffeic acid diminished the iso-butyrate concentration.
After 72 hours, we again observed significant results: the p-coumaric acid diminished the
concentration of acetate, while at the same time raising the concentration of iso-butyrate. All the
other ruminal parameters were not affected by the adding of these secondary plant metabolites.
CHAPTER IXTESTING THE PLANT SECONDARY METABOLITES ON THE FERMENTATION
PARAMETERS IN RUMINAL CULTURES ADDED PERIODICALLY IN VITRO
9.1 THE AIM AND THE OBJECTIVES OF THE EXPERIMENT
The aim of this experiment was to test two plant extracts represented by caffeic and p-coumaric
acid on gas production and on ruminal fermentation parameters added periodically in 12 mM
concentration in vitro. We added the plant secondary metabolites and tested the parameteres after 24,
48 and 72 hours.
9.2 RESULTS AND DISCUSSIONS
9.2.1 The effects of plant secondary metabolites added periodically in 12 mM concentration
The experiment in which we periodically added to the ruminal culture in a concentration of 12
mM the caffeic acid and p-coumaric acid, showed that after 24 h of incubation, the methane gas
xvi
emissions were reduced in the tests samples, and the effect was statistically significant. The control
sample had an average value of 4.87 ml/ml CH4, while the sample with caffeic acid had a value of 3.04
ml/100 ml methane, and the sample with p-coumaric acid had a value of 3.49 ml/100 ml CH4. After 24
hours, in the culture bottles was added a second dose of plant secondary metabollites in 12 mM, and the
measurments after 48 hours of incubation showed the same effect of diminuishing of methane
emissions. The next dose was added after 48 hours, and after 72 hours we observed that only the caffeic
acid had the the inhibiting effect on methane production.
This experiment showed that adding the phenolic acids periodically at the ruminal cultures, the
effect on methane gas production becomes stronger, producing its decrease. The other anlyzed gases
were not significantlly affected by caffeic and p-coumaric acid, with the exception of carbon dioxide
production which was significantly higher as compared to the control, after 48 hours of incubation. The
caffeic acid, after 24 hours, had a significant effect on iso-butyrate concentration, while the other
concentration of volatile fatty acids was not not significantly affected by its presence. After 24 hours of
incubation, we observed that the p-coumaric acid had an significantlly decreasing effect on acetate
concentration. The caffeic acid had significant effects on iso-butyrate concentration after 48 h of
incubation. After the third dose (72 h of incubation), it was observed that only the p-coumaric acid had
significant effects on acetate and iso-butyarte, the values beeing lower than control.
Following these experiments a conclusion was reached that these phenolic acids can adapt to
ruminal conditions and can be absorbed or used by microorganisms, methane-inhibiting effect being
lost quickly. However, the decrease of methane production was not accompanied by a major change in
volatile fatty acid concentrations and pH value. It is necessary that these plant secondary metabolites be
used in in vivo tests to accurately determine their effect on rumen fermentation parameters.
CHAPTER XTESTINFG OF IgY SPECIFIC IMMUNOGLOBULINS ON THE FERMENTATION
PARAMETERS IN RUMINAL CULTURES IN VITRO
10.1 THE AIM AND THE OBJECTIVES OF THE EXPERIMENT
The aim of this experiment was to test the IgY aviary antibodies on ruminal gas emissions.
Thus, the objective of the experiment was to measure the ruminal fermentation parameters (total gas
volume, gas composition, VFA concentrations, pH value) after 24, 48 and 72 hours of incubation of
ruminal cultures.
xvii
10.2 RESULTS AND DISCUSSIONS
10.2.1 The effects of IgY specific immunoglobulins on the fermentation parameters added insingle dose at ruminal cultures in vitro
The IgY antibodies had significant effects of decreasing the methane emissions after 24 hours
of incubation. Additionally, we observed a reduction of hydrogen production. The average value of
control sample was 4.87 ml/100 ml methane, and the test sample had a value of 3.77 ml/100 ml
methane. The single dose of antibodies does not have a significant effect on methane emissions after 48
hours of incubation, a decrease beeing only observed at hydrogen production. After 72 hours on
incubation, the effects were lost. The IgY antibodies had no significant effect on VFA profile after 24 h
of incubation. It was observed that after 48 h, the iso-butyrate concentration emitted by the test sample
was increased comparison to control sample. The antibodies increased significantlly the n-butyrate and
iso-valerate concentrations after 72 h of incubation and diminuished the iso-butyrate concentration. The
pH value was normal during the entire experiment, and we concluded that the IgY antibodies had no
effect on this parameter.
10.2.2 The effects of IgY specific immunoglobulins on the fermentation parameters addedperiodically at ruminal cultures in vitro
The IgY immunoglobulins were added as the caffeic and p-coumaric acids, periodically at the
ruminal cultures. Thus, it was observed that the antibodies had significant effects of decreasing the
methane emissions after 24 h of incubation. After 48 and 72 hours, the effect was lost. It was not
observed significant effects on hydrogen and carbon dioxide emissions during this experiment.
Significant effects were observed on n- and iso-butyrate concentrations, the antibodies decreased the n-
butyrate concentration and increased the iso-butyarate value after 24 hours of incubation. After the
second dose, were registered significant effects on propionate, n- and iso-butyrate concentrations. Thus,
the antibodies decreased the propionate and n-butyrate concentrations and increased the iso-butyrate
concentration. Significant effects were observed after 72 h of incubation, thereby, the antibodies
increased n-butyrate and iso-valerate concentrations and decreased iso-butyrate values.
After studying the literature, we were unable to identify any experiments that lasted 72 hours.
Thus, we concluded that our outcomes could be compared only to 48 hours, and it is possible that the
antibodies have been distorted during fermentation and lost activity after 48 hours. Following
xviii
molecular analyzes of the samples, in the ruminal fluid was observed that there were microorganisms
of the genus Methanobrevibacter, but could not be identified based on analyses carried out and the
species of methanogens. The antibodies were produced specifically to work against two
microorganisms of the genus Methanobrevibacter, and it is possible that these two species have not
been present in the studied ruminal fluid.
CHAPTER XIMOLECULAR CHARACTERIZATION OF RUMINAL METHANOGENS COMMUNITY
Methane production in the rumen may be affected by existing substrates necessary for the
rumen methanogenesis (especially hydrogen and carbon dioxide), the inhibition process of
methanogenesis and ruminal methanogens toxicity produced against it. Determining the rumen
methanogens microorganisms is required in a study which wants to analyze some compounds effects
on ruminal parameters.
11.1 THE AIM AND THE ONBJECTIVES OF THE EXPERIMENT
The aim of this experiment was to characterize the ruminal methanogens microorganisms at the
molecular level, both in control and in samples to which were added two of the phenolic acids
evaluated in other experiments as well as in avian antibody samples. Thus, in this experiment we used
samples of the culture bottles, to which were added caffeic and p-coumaric acids at the concentration of
12 mM, and avian IgY antibodies.
The objectives of this study were to isolate the genomic DNA from the ruminal fluid culture
bottles, to amplify the mcrA gene which is a marker for methanogens, and to identify these anaerobic
rumen microorganisms by a modern molecular analysis technique.
11.2 DNA ISOLATION FROM MIXED RUMINAL CULTURES
To identify and characterize the ruminal methanogens at a molecular level, DNA isolation
using an extraction kit was required as a first step. This was chosen based on previous results taken
after the first pre-experiment. The extraction kit was initially tested to evaluate outcomes on samples
taken from fresh ruminal fluid. After evaluating the results of this first experiment, the extraction kit
xix
was used on samples taken form culture bottles (control sample, sample with 12 mM caffeic acid,
sample with p-coumaric acid 12 mM and sample with IgY antibodies). The extraction kit used was
"NucleoSpin DNA Kit for Soil" from Macherey Nagel, which is designed specifically for the isolation
of high molecular weight genomic DNA from Gram positive or Gram negative microorganisms. It also
offers the possibility of using two lysis buffers that can be combined with chemical additives (Enhancer
SX), which guarantees a good purity for all the samples.
Following the isolation, genomic DNA was quantified using a NanoDrop ND1000
spectrophotometer, and after this step, the polymerase chain reaction was carried out to amplify the
gene of interest.
11.3 THE PCR REACTION
With the help of the polymerase chain reaction (PCR), the mcrA gene was amplified. The mcrA
gene encodes the methyl-coenzyme M reductase enzyme, which is important in the ruminal
methanogenesis process. The primers used in the amplification were the primers: mlas and mcrA-rev
(Steinberg and Regan, 2008) with a length of amplicons of 470-491 base pairs.
11.4 T-RFLP TECHNIQUE
Terminal Restriction Fragment Length Polymorphism (T-RFLP) is a molecular biology
technique for profiling of microbial communities based on the position of a restriction site closest to a
labelled end of an amplified gene. The method is based on digesting a mixture of PCR amplified
variants of a single gene using one or more restriction enzymes and detecting the size of each of the
individual resulting terminal fragments using a DNA sequencer. The result is a graph image where the
X axis represents the sizes of the fragment and the Y axis represents their fluorescence intensity.
11.5 RESULTS AND DISCUSSIONS
To identify the methanogenic microorganisms community in the rumen fluid, the T-RFLP
method was used. The samples subjected to this metode were taken after 24 hours of incubation of the
xx
ruminal cultures on which the caffeic and p-coumaric acids (12 mM), and the IgY antibodies were
added
11.5.1 The T-RFLP profiles for mcrA gene after the digestion with BstN enzyme
The restriction with BstN enzyme led to the identification for control samples of a 5 T-RF sites
that have been assigned, one T-RF with an abundance greater than 1% which could not be assigned and
8 T-RFs with an abundance under 1%, which also could not be assigned to any group of methanogens:
the T-RF 470 bp (Methanobrevibacter genus) with an abundance of 78.15%,, T-RF 473 bp
(Methanobacterium genus) with an abundance of 12.82 %, the T-RF 463 with an abundance of aprox.
3% and the T-RF 123 bp with an abundance of < 1%. For the samples with caffeic acid, it was
observed that the highest abundance is represented by the T-RF 470 bp corresponding to
Methanobrevibacter genus, which is 78.5 %. The Methanobacterium genus which was assigned to 473
bp sequence has an abundance of 11.41 %. For the 463 bp T-RF it was assigned also the
Methanobacterium genus, but the abundance of this t-Rf was lower, at about 3.40%. The terminal
restrction fragment which could not be attributed to any genus, the 462 bp (DCM-1), has in this
samples an abundance of approximately 5 %.The other assigend fragments, the 417 bp (DCM-1), 123
bp (Methanobacterium) and 94 bp (Methanoculleus), have an abundance of less than 1%.
Samples with p-coumaric acid 12 mM have the following sequences: 470 bp
(Methanobrevibacter genus) with an abundance of 76.25%, 473 bp (Methanobacterium genus) with
an abundance of 16.87 %, 463 bp (Methanobacterium genus) with an abundance of approx. 3%, and
the T-RF 123 bp and the T-RF 417 bp with an abundance of less than 1%.
The 470 bp T-RF (Methanobrevibacter genus) has an abundance of 78.25% for the samples
with IgY antibodies, which is the predominant genus. The T-RF 473bp has an abundance of 12% ant
the T-RF 463 has an abundance of approx. 3%. The other fragments have an abundance less tha 1 %
(417 bp, 123 bp, 94 bp) and the fragment that could not be assigned (462 bp T-RF) has an abundance
of more than 5%.
xxi
11.5.3 The T-RFLP profiles for mcrA gene after the digestion with MwoI enzyme
With the help of the T-RFLP results obtained after the restriction with MwoI enzyme, it could
be attributed to gender four terminal restriction fragments, from which only two have an abundance
greater than 1% (210 bp and 228 bp). Another 12 fragments, with an abundance less than 1 %, could
not be attributed to any kind of genus from the clone library that we used. The most abundant T-RF it
was the one at 210 bp which was assigned to Methanobrevibacter genus, so the replicates for the
control samples have an abundance of 20.77 % , an abundance of 20.50 % for caffeic acid samples and
for p-coumaric acid samples the abundance was 25 %. The IgY antibodies samples have an abundance
of about 24%.
The 228 bp fragment that was attributed to DCM-1 Archaeon had an abundance of over 10%
for all samples. The greatest abundance was observed for p-coumaric acid with 14.13 %, followed by
control samples with an abundance of 11.32 %. The samples with IgY antibodies showed an abundance
of 11%, while caffeic acid samples have an abundance of 10.75 %. With this enzyme it was posible to
assigne only one fragment to Methanobacterium genus. The abundance of this fragment (438 bp),
however, is less than 1% for all samples. For Methanobrevibacter genus, it was assigned another
fragment, 444 bp, but this T-RF has an abundance less than 1%. A greater abundance has the 311 bp
fragment, which could not be assigned to any genus. This T-RF has an abundance of approx. 25.5 %
for the control samples, caffeic acid samples and IgY samples, and approx. 17.5 % for p-coumaric
samples. The 443 bp fragment has an abundance between 6.5 and 9.9 %, but even this one could not be
assigned on the basis of the clone library.
CHAPTER XIIGENERAL CONCLUSIONS
After all the testing done for this PhD thesis, we concluded that by adding in single dose the
caffeic acid and p-coumaric acid in 12 mM concentration to ruminal cultures in vitro, after 24 hours of
incubation, the methane gas reduces significantly. The caffeic acid showed the same effect after 48
hours of incubation. Also adding the phenolic acids periodically, we observed significant results of
decreasing the ruminal methane after 24 and 48 hours of incubation.
We observed that the VFA concentrations did not undergo any major modifcations as a result of
adding the phenolic acids to the ruminal cultures. The effects the phenolic acids on the concentration of
xxii
VFA is based on the anti-microbian properties of these extracts. The majority of results showed that
VFA values were smaller in the test samples as compared with the control samples, but not
significantly. The small difference between the test and the control samples indicates the posibility of
utilizing these extracts as fermentation substrata. These extracts are the trans-cinnamic acid and
catechin hydrate in 6 mM concentration.
The IgY aviary antibodies had significant effects on the emissions of methane gas only after 24
hours of incubation. We did not observed statistically significant effects on any of the other parameters
tested. After researching the literature in this domain, we concluded that our results can be compared
only for up to 48 hours, and it is posible that the antibodies were destroid during the fermentation
process, as they lost some of their mobility after 48 hours. After molecular analyses of the samples, we
observed that inside the ruminal liquid there were microorganisms of the Methanobrevibacter genus,
but we could not properly identify the methanogen species. The antibodies were produced for a specific
purpose: to fight against two microorganisms from the Methanobrevibacter genus, and it is possible
that tese two species were not presented in the ruminal liquid that we tested.
The results of our experiments in these PhD thesis showed that by adding certain secondary
plant metabolites to the ruminal cultures, we had a significant reduction of the methane gas after 24 or
48 hours of incubation. Adding the caffeic acid in 12 mM concentration had yielded the best result of
reducing the methane gas, as this acid showed significant effects after 48 hours as well. The next plant
metabolite utilized that had significant reduction effects is the p-coumaric acid in 12 mM concentration
which showed effects after 24 hours of incubation.
Significant reduction effects of the methane gas were presented by the aviary IgY antibodies
after 24 hours of incubation.
We also observed significant reduction effects of the H2 and CO2 emissions and on the VFA
concentration, specifcally on the acetate, propionate and iso-butyrate.
Adding these secondary plant metabolites and aviary IgY antibodies did not affect the pH level,
which was at a stable level in the ruminal liquid. We did not observe significant effects on any of the
tested parameters when the secondary plant metabolites were added in 6 mM concentration; the trans-
cinnamic acid and the catechin hydrate were eliminated after our first experiment for this particular
reason.
With the help of T-RFLP analysis with which we wanted to identify the members of ruminal
methanogens from the ruminal fluid it was possible to identify four types of methanogens, which are:
xxiii
Methanobrevibacter spp., Methanobacterium spp., Methanoculleus spp., and DCM 1 Archaeon. It
turned out that the mcrA gene is a marker for methanogens and its amplification was a good choice.
Also, the enzyme with the best results was the BstNI enzyme.
SELECTIVE BIBLIOGRAPHY
1. BERGMAN E.N, REID R.S., MURRAY M.G., BROCKWAY J.M., WHITELAW F.G., 1965,
Interconversions and production of volatile fatty acids in the sheep rumen, Biochem. J, 97/53.
2. BRYANT M. P., 1959, Bacterial species of the rumen. Bacteriol. Rev., 23:125-153.
3. CARULLA J.E., KREUZER M., MACHMÜLLER A., HESS H.D., 2005, Supplementation of
Acacia mearnsii tannins decreases methanogenesis and urinary nitrogen in forage-fed sheep,
Australian Journal of Agricultural research, 56:961–970.
4. COOK S.R., MAITI P.K., CHAVEZ A.V., BENCHAAR C., BEAUCHEMIN K.A.,
MCALLISTER T.A., 2008, Avian (IgY) anti-methanogen antibodies for reducing ruminal
methane production: in vitro assessment of their effects, Australian Journal of Experimental
Agriculture, 48:260-264.
5. GIUBURUNCĂ MIHAELA, CRISTE ADRIANA, COCAN D., CONSTANTINESCU R.,
RĂDUCU CAMELIA, MIREŞAN VIOARA, 2014, Effects of plant secondary metabolites on
methane production and fermentation parameters in in vitro ruminal cultures, Scientific Papers:
Animal Science and Biotechnologies, 47-2:78-82.
6. GIUBURUNCĂ MIHAELA, CRISTE ADRIANA, MIREŞAN VIOARA, 2015, Effects of p-
coumaric acid on ruminal fermentation parameters in in vitro ruminal cultures, Bulletin of
UASVM Cluj-Napoca, Animal science and Biotech., 72 (1).
7. GOEL G., PUNIYA A.K., AGUILAR C.N., SINGH K., 2005, Interaction of gut microflora
with tannins in feeds, Naturwissenschaften, 92:497-503 .
8. HASNA EL GHARRAS, 2009, Polyphenols: food sources, properties and applications–a
review, International Journal of Food Science & Technology, 44:2512–2518.
xxiv
9. JANSSEN P.H., 2010, Influence of hydrogen on rumen methane formation and fermentation
balances through microbial growth kinetics and fermentation thermodynamics, Anim. Feed. Sci.
Tech., 160:1-22.
10. JAYANEGARA A., TOGTOKHBAYAR N, MAKKAR HARINDER P.S., BECKER K., 2010,
Tannins determined by various methods as predictors of methane production reduction potential
of plants by an in vitro rumen fermentation system, Animal Feed Science and Technology
150:230–237.
11. LASCANO C.E. ȘI CARDENAS E., 2010, Alternatives for methane emissions mitigation in
livestock systems, R. Bras. Zootec., 39:175-182.
12. MARCQ C., THÉWIS A., PORTETELLE D., BECKERS Y., 2010, Keep bacteria under
control: Dietary modulation of gut microflora in farm animals by use of hen egg yolk
antibodies, (http://hdl.handle.net/2268/81363).
13. MARTIN C., MORGAVI D.P., DOREAU M., 2009, Methane mitigation in ruminants: from
microbe to the farm scale, Animal, 1-15.
14. MIREŞAN VIOARA AND MIREŞAN E., 1997, Producerea cărnii de tineret ovin, Editura
Genesis, Cluj-Napoca.
15. MIREȘAN VIOARA, ERSEK ADEL AND RĂDUCU CAMELIA, 2003, Fiziologia animalelor
domestice, Editura Risoprint, Cluj-Napoca.
16. MORAN J., 2005, Tropical dairy farming: feeding management for small holder dairy farmers
in the humid tropics, Editura Landlinks.
17. PINARES C.S., ULYATT M.J., WAGHORN G.C., LASSEY K.R., BARRY T.N., HOLMES
C.W., JOHNSON D.E., 2003c, Methane emission by alpaca and sheep fed on lucerne hay or
grazed on pastures of perennial ryegrass/white clover or birdsfoot trefoil. Journal of
Agricultural Science 140:215–226.
18. PUCHALA R, MIN BR, GOETSCH A.L., SAHLU T., 2005, The effect of a condensed tannin-
containing forage on methane emission by goats, Journal of Animal Science 83:182–186.
19. SEJIAN V., LAL R., LAKRITZ J., EZEJI T., 2011, Measurement and prediction of enteric
methane emission, Measurement and prediction of enteric methane emission, Int. J.
Biometeorol, 55:1-16.
20. SIROHI S.K., PANDEY N., SINGH B., PUNYA A.K., 2010, Rumen methanogens: a review,
Indian J. Microbiol., 50:253-262.
xxv
21. WRIGHT A.D.G., KENNEDY P., O’NEILL C.J., TOOVEY A.F., POPOVSKI S., REA S.M.,
PIMM C.L., KLEIN L., 2004, Reducing methane emissions in sheep by immunization against
rumen methanogens, Vaccine, 22:3976-3985.
22. ZORZOLIU C. AND ZORZOLIU LIVIA, 1992, Estimarea emisiei anuale de metan la bovinele
din România în perioada 1938-1989. Analiză ecologică şi perspectivă., Simpozion “Factorii de
mediu, producţia şi sănătatea taurinelor”, Cluj-Napoca.
23. ***www.epa.gov
24. ***www.ncbi.nlm.nih.gov