University of Veterinary Medicine Hanover
The effects of a subacute rumen acidosis and the subsequent recovery process on fermentation patterns and the microbial
community using the Rumen Simulation Technique
Inaugural-Dissertation
in fulfillment of the requirements of the degree of Doctor of Veterinary Medicine
-Doctor medicinae veterinariae-
(Dr. med. vet.)
submitted by
Theresa Wilhelmina Orton, née Maasjost
Vechta
Hannover 2019
University of Veterinary Medicine, Hanover Institute for Physiology and Cell Biology
Melanie Eger, Ph.D.
University of Veterinary Medicine, Hanover Institute for Physiology and Cell Biology
1st Referee: Prof. Dr. Gerhard Breves
2nd Referee: Prof. Dr. Martina Hoedemaker, Ph.D.
Day of the oral examination: 05.11.2019
Meiner Familie
T Orton, K Rohn, G Breves, M Eger, 2019
Alterations in fermentation parameters during and after induction of a subacute rumen acidosis in the rumen simulation technique
T Orton, M Eger, B Pinior, F Roch, B Zwirzit, G Breves, S U Wetzels
Analyzing the impact of an in vitro subacute rumen acidosis on the bacterial community and the recovery process applying amplicon Illumina MiSeq and PacBio sequencing
Parts of this thesis have been presented at a conference:
T Maasjost, M Eger, G Breves
Effects of different concentrate levels and buffer compositions on the
induction of a subacute acidosis by applying the Rumen Simulation Technique
73rd Conference of the Society of Nutrition Physiology, 13th – 15th March, 2019 in Göttingen
Abstract published in the Proceedings of the Society of Nutrition Physiology Volume 28, 2019;; ISBN 978-3-7690-4112-5, p. 119.
G Breves, S U Wetzels
The bacterial community dynamics in a subacute ruminal acidosis evaluated by Rumen Simulation Technique (RUSITEC)
International Symposium on Ruminant Physiology, 3rd – 6th September, 2019 in Leipzig
Abstract published in the Proceedings of the XIIIth International Symposium on Ruminant Physiology (ISRP 2019), Advances in Animal Biosciences, ISSN 2040-
4700, p. 632.
Introduction ... 11
1 Review of literature ... 13
1.1 Degradation of feed and pH regulation in the rumen ... 13
1.2 Rumen acidosis ... 20
1.3 Bacterial alterations during subacute rumen acidosis ... 23
1.4 In vivo models for subacute rumen acidosis ... 26
1.5 In vitro models for subacute rumen acidosis ... 28
2 Material and methods ... 31
2.1 Animals ... 31
2.2 Experimental set up: The Rumen Simulation Technique ... 31
2.3 Experimental design and sampling scheme ... 34
2.4 Measurement of fermentation parameters ... 35
2.4.1 pH and redox potential ... 35
2.4.2 Lactate ... 35
2.4.3 Ammonia-N ... 36
2.4.4 Short chained fatty acids ... 36
2.4.5 Degradation of organic matter ... 37
2.5 Microbial isolation and DNA preparation ... 38
2.5.1 Sequencing of the bacterial community ... 39
2.5.2 Illumina MiSeq ... 39
2.5.3 PacBio ... 40
3 Results in form of two manuscripts ... 42
3.1 Alterations in fermentation parameters during and after induction of a subacute rumen acidosis in the rumen simulation technique ... 42
community and the recovery process applying amplicon Illumina MiSeq and
PacBio sequencing ... 72
4 Discussion ... 127
4.1 Material and methods ... 127
4.1.1 Donor animals and inoculum ... 127
4.1.2 The Rusitec system as an in vitro model ... 127
4.1.3 Induction of subacute acidosis in the Rusitec model compared to in vivo methods ... 130
4.1.4 DNA extraction procedure ... 133
4.1.5 The application of Illumina MiSeq and PacBio sequencing in rumen research ... 134
4.2 Results of the biochemical and microbial analysis ... 137
4.2.1 Acidosis induction affects fermentation patterns and bacterial abundances ... 137
4.2.2 The roughage-to-concentrate ratio influences the ruminal fermentation pattern ... 141
4.2.3 Fermentation pattern and bacterial abundance is able to recover from acidosis ... 144
6 Summary ... 147
7 Zusammenfassung ... 150
8 References ... 153
9 Appendix ... 174
9.1 Buffer solutions for pH maintenance in the Rusitec ... 174
9.2 Methylcellulose solution for the detachment of solid associated microorganisms ... 174
9.3 Ammonia-N measurement ... 174
10 Danksagung ... 175
Figures
Figure 1 The Rusitec model ... 33 Figure 2 Experimental design and sampling schedule ... 34
°C degree Celsius
µl micro liter AI acidosis I buffer AII acidosis II buffer AP acidosis period
Aqua dest. aqua destillata (destilled water) ARA acute rumen acidosis
ASV amplicon sequence variant CaCl calcium chloride
cm centimeter
CO2 carbon dioxide CP I first control period CP II second control period
d day
DNA deoxyribonucleic acid
g gram
g centrifugal acceleration expressed as a multiple of gravity (gn = 9.80665 m ∙ s -2)
h hour
H hydrogen
H2O water
HCl hydrochloric acid HCO3 bicarbonate
KCl potassium chloride
kg kilogram
LAM liquid associated microorganisms
M molar
min minutes
ml milliliter
mM Millimolar
mm2 square millimeter mmol millimole
N nitrogen
Na+ sodium
NaH2PO4 sodium dihydrogen phosphate Na2HPO4 disodium hydrogen phosphate NaCl sodium chloride
NaHCO3 sodium bicarbonate NaOH sodium hydroxide
NGS next generation sequencing NH3-N ammonia N
NH4Cl ammonium chloride
nm nanometer
OM organic matter
OTU operational taxonomic unit p probability value
PCR polymerase chain reaction RNA ribonucleic acid
rpm rounds per minute
SAM solid associated microorganisms SARA subacute rumen acidosis
SCFA short chained fatty acids SD standard deviation SDS sodium dodecyl sulfate SEM standard error of mean
SMRT single-molecule real-time sequencing ST standard buffer
ZMW zero-mode wave guides
Introduction
In the past decades, cattle farming has changed dramatically. To satisfy the world’s increasing population and the demand of high-quality animal protein, production efficiency has increased by 54% in dairy and 45% in beef production (Pitta et al., 2018;;
USDA_NASS, 2014). While the number of dairy farms has continuously declined, the individual milk yield of each animal increased significantly (Capper et al., 2009). The elevated demand can only be met through genetic selection focusing on a high yielding production rate, at the expense of longevity and general health (Oltenacu and Broom, 2010). The productivity of the individual animal is dependent on the microbial community in the rumen, which enables the animal to digest and breakdown cellulose and plant material (Mizrahi, 2013). The rumen microorganisms supply the animal with energy in form of short chained fatty acids (SCFA) and protein (Barcroft et al., 1944, France and Dijkstra, 2005). One of the most common disorders in dairy cattle farming is rumen acidosis (Nagaraja and Lechtenberg, 2007), which describes a fermentative dysfunction based on an impaired rumen pH regulation. This mostly affects high yielding dairy cows, especially during early lactation and beef cattle in the finishing period (Nocek, 1997, Kleen et al., 2003). During this time, the animals experience a dietary change. Beef cattle are fed intensively with concentrate to enhance meat production, while the onset of lactation demands an increased energy supply. The transition from low to high grain diets is challenging for the rumen mucosa and the rumen microbial community and therefore sufficient time for adjustment is required. An increasing amount of dietary concentrate enhances the accumulation of SCFA in the rumen, which lead to a decrease in ruminal pH (Krause and Oetzel, 2005).
Maladaptation enhances the probability of rumen acidosis (Dirksen et al., 1984).
Despite of certain predispositions, all ruminants can experience rumen acidosis (Enemark et al., 2002). Several sequelae are reported to be associated with rumen acidosis, such as laminitis, diarrhea, and ruminitis (Stone, 2004). Furthermore, the reduced milk yield and a diminished amount of milk fat have significant financial implications. Due to the major effects of the rumen acidosis on animal health and wellbeing, and furthermore, the great financial and economic consequences, this fermentative dysfunction has been a major topic of research for the past decades. With the present work, we aimed to provide an in vitro model to mimic subacute acidosis under laboratory conditions. We investigated the impact of an acidotic challenge in an in vitro model, firstly, observed changes in the ruminal fermentation pattern, and secondly alterations in the bacterial population. Furthermore, we analyzed the recovery of fermentation patterns and the bacterial population within this in vitro model. The present work aims to provide an in vitro system to participate in the numerical reduction of animal testing in primary research and provide new possibilities of testing feed additives improving acidosis treatment and prevention strategies. Furthermore, we aimed to improve the knowledge about the bacterial population dynamics within this in vitro model and evaluated the usability of two next generation sequencing approaches.
1 Review of literature
1.1 Degradation of feed and pH regulation in the rumen
Ruminants are very well adapted to digest plant material, which is rich in fiber and low in energy. Therefore, the ruminal flora and the host ruminant form a strong symbiotic relationship. A large group of bacteria, protozoa and fungi decompose plant material, which the host animal alone would not be capable of breaking down (McCann et al., 2016). The majority of the ruminant’s energy requirements are met by microbial fermentation (Byrant, 1970). Ammonia and peptides, as much as fiber and carbohydrates deliver the majority of the energy (Pitta et al., 2018). This includes neutral detergent insoluble fiber polysaccharides, like cellulose and hemicellulose.
Furthermore, the microbial community digests non-fiber carbohydrates, like sugars, starch and water-soluble carbohydrates (pectin, b-glucans, galactans). Side products include carbon dioxide and methane. Gasses escape the rumen through eructation, which implies an energy loss due to methane of more than 12% (Pitta et al., 2018).
The retention time within the forestomach is dependent on the forage to concentrate ratio (Bartocci et al., 1997) and the particle size (Welch, 1986). The retention time varies between 18 h (Poppi et al., 1981) and 72 h, and is of major importance for the degradation of feed particles, but also for the maintenance of the microbial diversity.
Due to the non-fastidious nature of protozoa and fungi, both are especially dependent on long retention times. With generation times of 5 to 14 h for ruminal protozoa and up to 30 h for fungal communities shortened retention times may lead to a rapid deletion of these organisms (McAllister et al., 1994).
Even though Protozoa species maintain up to 50% of the microbial mass in the rumen, their role within the community has not yet been completely elucidated (Newbold et al.,
2015, Levy and Jami, 2018). There are two main groups within the ciliate population, which differ morphologically and also in their substrate preferences. Holotricha, with almost uniformal somatic cilia mostly degrade soluble substances, whereas entodinomorphoid protozoa are characterized by a firm pellicle and the possession of cilia on the peristome. These ciliates mainly digest smaller, solid particles (Belanche et al., 2015a). Protozoa gain particular significance in the ruminal ecosystem, as they participate in the ruminal pH maintenance (Lee et al., 2000). Protozoa restrict the availability of starch and glucose molecules from rapid bacterial fermentation. The ciliates ferment substrates slowly and release them into the rumen at a much reduced rate (Newbold et al., 1989, Williams and Coleman, 1997). Contrarily to the fast substrate conversion, the protozoal fermentation leads to a steady production rate of SCFA (Michałowski, 1987) and therefore enhances ruminal pH stability (Newbold et al., 2015). A slow increase of the concentrate ratio in the feed allows the ciliate population to adapt to the enhanced number of fermentable substrates, without being overgrown by the bacterial community (Nagaraja et al., 1986, Nagaraja and Titgemeyer, 2007). The role of protozoa regarding the fermentation patterns and interactions between archaea and fungi is still a topic of interest.
Like the protozoan community, the fungal group belongs to the slow growing microbes in the rumen and is of significant importance in cellulose degradation. Fungi are dependent on a gradual breakdown of substrates and a long ruminal retention time to be able to contribute productively to the rumen digestion (McAllister et al., 1994). The ruminal fungi are able to attach to the intact surface of feed particles by destroying the outer protection barrier with hypha, contrarily to the bacterial fraction, which is dependent on damages and porous structures in order to bind to particles. Therefore,
fungi contribute significantly to the attachment of bacteria on feed particles and assist in the bacterial colonization of fiber (Lee et al., 2000). The abundance of fungi within the rumen is determined by the roughage ratio (Gruninger et al., 2014).
The bacterial community is by far the most diverse group within the ruminal flora. This group can be subdivided into three major parts. Firstly, there are liquid associated microorganisms, which can be found mainly in the fluid fraction of the ruminal content.
Secondly, a major group is associated with the solid phase, where bacteria are attached to feed particles. Within the solid associated microorganisms one fraction attaches tighter to particles as other bacteria. Loosely attached bacteria can be removed by gentle washing, whereas the firmly attached bacteria remain on the particle’s surface (McAllister et al., 1994). Thirdly, a smaller group of epimural bacteria bonds to the rumen wall (Cheng et al., 1979, Sadet et al., 2007). This last group forms the smallest part of the bacterial biomass (Sadet et al., 2007). Sadet et al. (2007) analyzed the epimural community of lambs and the impact of feed alterations on the diversity on this specific bacterial group. The authors could not determine any feed associated influences;; however, the individual animal had great impact on the diversity of the epimural flora. The individual alterations are said to be due to the co-dependent multifactorial relationship between the host animal and the epimural community. Chen et al. (2011) confirmed the individual influence;; however, authors did also imply an impact of feed. Reasons for the variation among studies remain under discussion.
Epimural bacteria are less involved in the fermentation of SCFA, but have major importance to maintain the ruminal anaerobic conditions, as they are able to bind oxygen. Furthermore, the epimural flora is involved in urea metabolism and digests devitalized epithelial cells (Cheng et al., 1979).
For degradation of structural plant material, it is obligatory for the microbial flora to attach to feed particles. Therefore, bacteria have to overcome certain protective barriers. Layers of cuticle, which may contain up to 24% silica, protect grains and forages. The cuticle surface enhances the rigidity and impedes digestion.
Nevertheless, bacteria penetrate this surface via stoma, lenticels, or damaged areas and start to colonize and digest from the inside out (Cheng et al., 1991, McAllister et al., 1994). This is initially done by primary colonizers of the liquid associated flora.
Using binding proteins and glycocalices, these microorganisms attach to the particle surface and enzymatically degrade insoluble substrates. After fermentation, soluble nutrients are released into the liquid fraction. In a second step, the solid associated bacteria are able to attach to the gylcocalicae of the primary colonizers (McAllister et al., 1994). This attachment forms a biofilm, which protects the particle associated microflora from antibacterial substances and potential predators, like protozoa (Newbold et al., 1989, McAllister et al., 1994). Within this microclimate, electrons and nutrients can be transported safely and finally degradation products like SCFA can be released into the rumen. When the cellular walls of the feed particles have been completely degraded, bacteria attach to the next feed particle. Non- or partly digested particles are eventually transported further down the digestive system (McAllister et al., 1994). The particle associated bacterial population is estimated to account for 70 – 80% of the bacterial mass (Craig et al., 1987, McAllister et al., 1994). The primary studied cellulolytic bacteria are Fibrobacter succinogenes, Ruminococcus flavefaciens and Ruminococcus albus (Weimer, 1992, McAllister et al., 1994, Weimer, 1996, Oss et al., 2016). The mean retention time of feed particles in the solid phase is higher compared to the fluid fraction. Furthermore, the enzymatic activity is significantly higher
in the solid phase (McAllister et al., 1994). The enzymatic activity in cell free fluid rumen samples is reported to decrease rapidly, which indicates a fast inactivation of enzymes produced by fluid associated bacteria. This observation leads to the presumption, that the solid associated bacteria are responsible for the major part of feed digestion (McAllister et al., 1994). The microbial degradation of carbohydrates mainly results in the production of SCFA, of which acetate, propionate, and butyrate are of major importance to the animal. The majority of the SCFA are absorbed through the rumen wall, but also in the further digestive tract.
The ruminant uses acetate and butyrate primarily as an energy source, resulting from oxidation via the citric acid cycle (France and Dijkstra, 2005). Acetate is the main source for lipogenesis, which is reflected in the milk fat production in the mammary gland (Weimer et al., 2010, Weimer et al., 2017). Butyrate also promotes ruminal epithelial cell proliferation (Sakata and Tamate, 1978). As the net glucose uptake in the rumen and intestinal tract is relatively low in ruminants (Roe et al., 1966), propionate is a major source for glucose production. Propionate is absorbed majorly through the rumen wall and contributes significantly to gluconeogenesis in the liver (Bergman et al., 1966).
The total SCFA concentration in the rumen varies between 70 and 130 mM, resulting from production and absorption processes. The concentration of individual SCFA is referred to as the fermentation pattern (France and Dijkstra, 2005). The composition and total appearance of the individual SCFA are influenced by various factors, such as the source of carbohydrates, pretreatment of feed and feeding portions (Strobel and Russell, 1986, Lettat et al., 2010). Feeding high roughage rations generally results in a high production of acetate, whereas high starch ratios lead to an increased propionic
acid production (Sutton et al., 2003). Butyrate production is commonly favored by the fermentation of soluble carbohydrates (Enemark et al., 2002, Weiss et al., 2017).
Feeding a high forage-ration, the acetate:propionate:butyrate ratio generally ranges around 70:20:10 (France and Dijkstra, 2005). Under physiological feeding conditions, the rumen pH varies between 7.1 and 6.0 (Shi and Weimer, 1992). After feed intake SCFA accumulate in the rumen and reduce the pH for several hours (Allen, 1997, Soto-
Navarro et al., 2000b). Shifts of 0.1 to 1.0 pH units within a 24 h period are common (Oetzel, 2007). To encounter the decreasing ruminal pH, acids are then mainly absorbed through the rumen wall or transported further along the gastrointestinal tract.
Furthermore, ruminal buffer mechanisms neutralize accumulating acids to maintain the physiological pH range. The epithelial cells provide three main mechanisms to maintain the ruminal milieu and to protect the intracellular pH from decreasing. The ruminal mucosa absorbs both forms of SCFA, the ionized and protonated form (Kramer et al., 1996). With pKa (the pH point of maximum buffering) values of 4.8, SCFA are largely abundant in the dissociated form in the rumen. Based on the Henderson-Hasselbalch equation, the appearance of dissociated and undissociated acids is based on an equilibrium reaction. A decreasing rumen pH is associated with an enhanced proportion of undissociated SCFA and equally increases the rate of diffusion (France and Dijkstra, 2005). The passive absorption is a very fast process, which is beneficial when a high amount of SCFA accumulate in the rumen after feed intake (Kramer et al., 1996). The ruminal wall provides two apical transport mechanisms for enhancing SCFA absorption and protecting the ruminal and intracellular pH milieu: a Na+/H+ exchanger and a bicarbonate importing system (Schweigel et al., 2000). In pH ranges above pH 6.0, SCFA are mainly present in their ionized form, due to their pKa of 4.8.
Located on the luminal side of the epithelium, Na+/H+-exchanger release one H+ into the rumen in exchange for one Na+ ion, which is transported into the cell. The free protons create a microclimate around the rumen wall and convert ionized SCFA into the undissociated form. Protonated SCFA are then passively absorbed by the rumen epithelium. Due to the electrical gradient, the uptake of ionized SCFA is accompanied with either anion secretion or cation absorption. Bicarbonate secretion is the driving force for the SCFA uptake. Within the ruminal epithelium bicarbonate is converted from carbon dioxide by the carbonic anhydrase. Therefore, the ruminal epithelia cells provide bicarbonate for a second transport mechanism, which releases HCO3- into the rumen in exchange of dissociated SCFA (Gäbel et al., 2002, Dijkstra et al., 2012).
HCO3- is one of the most effective buffers in the rumen. With a ruminal concentration of approximately 120 mM, HCO3- neutralizes up to 50 – 60% of the ruminal accumulated acids (Dijkstra et al., 2012). Bicarbonate is able to neutralize H+ protons, by binding and transformation into carbonic acid. The carbon acid dissociates to CO2 and H2O, of which CO2 escapes the forestomach by ruminal eructation.
The chain length of the individual SCFA has an impact on resorption and due to this the absorption of the three main SCFA increases in the order acetate < propionate <
butyrate (Gäbel et al., 1991). A slow increase of easily fermentable carbohydrates, and therefore a stepwise increase of acids in the rumen leads to a proliferation of rumen epithelia cells, increases the growth of ruminal papillae and enhances the blood supply.
These morphological processes improve the actual resorption capacity and may lead to an up to four-fold increase of the net absorption/disappearance rate of SCFA (Dirksen et al., 1984). Dirksen et al. (1984) implied an adaptation time of at least four
weeks for the optimal adjustment of the ruminal mucosa to an enhanced concentrate ration.
Additionally to the buffering mechanisms inside the forestomach, the ruminant’s saliva contributes significantly to the pH maintenance, as it is very rich in HCO3- and phosphate (McDougall, 1948). Up to half of the ruminal bicarbonate is derived from saliva (Owens et al., 1998b). The phosphate buffering function differs from bicarbonate, as phosphate is only removed by further passage. At pH 6.0 more than 94% of the phosphate is complexed as dihydrogen phosphate and leaves the rumen in the liquid phase (Allen et al., 2006). Saliva has a pH of about 8.4 and is a very effective buffer for pH values lower than pH 6.0 (McDougall, 1948). Many studies claim that a high structural roughage feeding increases the chewing time and simultaneously enhances the buffer capacity in the rumen (Beauchemin, 1991, Allen, 1997, Beauchemin et al., 2003). However, the impact of feed structure on the pH regulation is discussed controversially. Jiang et al. (2017) observed an increased production of saliva when animals were fed with roughage, however, the daily total amount of saliva did not significantly increase. The authors conclude that the amount of structure within the ration has no direct impact on the total saliva production and therefore, it has no impact on the ruminal pH development. Maekawa et al. (2002b) supported this statement, and reported a decreasing saliva production in between chewing time and rumination period.
1.2 Rumen acidosis
Forages and concentrates differ in their proportion of structural and non-structural carbohydrates, nitrogenous substances and lipids (Pitta et al., 2018). A simple forage
diet can meet all the animal’s nutritional needs, however, when it comes to productivity and milk yield, an energy supplementation in form of concentrate feeding is needed (Hills et al., 2015). A highly energized diet, which is rich in easily fermentable carbohydrates but lacks fiber and structure, leads to an enhanced production of SCFA in the forestomach (Beauchemin, 2007). When these acids accumulate in the rumen and the balance between neutralization and acid production is out of equilibrium, the imbalance leads to a reduction of the ruminal pH below physiological thresholds (Ash and Dobson, 1963). Allen (1997) reports a very sensitive reaction of pH to feed intake.
The reduction of ruminal pH below critical thresholds over a certain time period (Penner et al., 2007, Khafipour et al., 2009b) is referred to as rumen acidosis. An increased presence of protons during acidosis overstrains the resorption capacity of the epithelium and cannot be eliminated. This leads to functional alterations within the epithelium (Liu et al., 2013, Schwaiger et al., 2013b).
Ruminal acidosis may appear in an acute and a subacute form. Acute rumen acidosis (ARA) describes a fermentative dysfunction, where the ruminal pH decreases below pH 5.2 (Penner et al., 2007) or < pH 5.0 (Enemark et al., 2002). The low pH values favor the growth and activity of lactate-producing microorganisms, which leads to an increasing ruminal lactate concentration. In pH ranges above pH 5.5, lactate does not accumulate in the rumen (Slyter, 1976) as the production is modulated by the activity of lactate utilizing bacteria, e.g. Megasphera elsdenii and others (Arik et al., 2019).
These bacteria use lactate to produce SCFA, however, their growth and activity are impaired below pH 5.0 (Arik et al., 2019). Therefore, a subacute rumen acidosis may lead to a severe lactic acidosis (Nagaraja and Titgemeyer, 2007). The enhanced lactate production consequently supports a decreasing pH trend (Owens et al., 1998),
as it’s pKa is significantly lower than SCFA (3.8 vs. 4.8, respectively). The clinical appearance of the acute acidosis can be dramatic. Severe acidosis leads to a rapid deterioration of the general condition. While anorexia and a decreasing milk production are signs of a mild form of ARA, a severe outbreak can induce recumbency, coma and finally death within 8 to 10 h (Bramley et al., 2005).
In contrast, during a subacute rumen acidosis (SARA) the decrease in rumen pH appears less severe. Some authors define SARA as a decrease between pH 5.5 to 5.0 (Enemark et al., 2002) or between pH 5.6 and 5.2 (Cooper et al., 1998). However, under physiological feeding conditions, the ruminal pH decreases for several hours postprandial until the accumulated SCFA are absorbed and neutralized (Beauchemin, 2007, Dijkstra et al., 2012). Therefore, the time spent below certain thresholds has been used to identify SARA. Zebeli et al. (2008) define SARA as a time of more than 300 min below pH 5.8, whereas Khafipour et al. (2009a) use the threshold of 5.6 for more than 180 min. Contrarily to ARA, the lactate concentration in the rumen remains low in subacute acidosis. This is either due to the enhanced lactate utilization (Long et al., 2014) or results from a reduced growth and abundance of lactate producing bacteria, compared to ARA (Krause and Oetzel, 2005). Mostly, SARA occurs individually and recurrent over a longer period. Signs may be laminitis, diarrhea, a decreasing milk production and a reduced feed intake (Nocek, 1997, Enemark et al., 2002, Kleen et al., 2003). Repetitive acidotic bouts irritate the rumen mucosa and increase the susceptibility for mucosal damages and dysfunctions. Consequently, parakeratosis and ruminitis can occur (Krehbiel et al., 1995, Kleen et al., 2003). Gram-
negative bacteria are very sensitive towards lower pH values. Cellular death and cell lysis of these bacteria release membranous lipopolysaccharides, which cause further
damage to the integrity of the ruminal epithelium (Mao et al., 2013). These defects lead to an impaired SCFA uptake, and mucosal lesions may provide entry for pathogens, like Fusobacterium necrophorum or Trueperella pyogenes. The ruminitis liver abscess complex and laminitis are common pathological sequelae (Nocek, 1997, Svensson and Bergsten, 1997). Nevertheless, in contrast to the acute rumen acidosis obvious clinical signs of SARA are generally missing or harder to detect (Cooper et al., 1998).
Subsequently, the number of subclinically affected animals remains high. Moreover, the concomitant diseases and the reduced productivity are of major economic concern for the farmer. In Germany and the Netherlands, approximately 11% of the early and 18% of the mid lactating animals are affected by SARA (Kleen et al., 2004). In a recent study, Stefańska et al. (2016) analyzed the prevalence of SARA in 213 polish dairy herds. In more than 44% of the herds more than 25% of the animals suffered from SARA.
1.3 Bacterial alterations during subacute rumen acidosis
In order to be able to colonize feed particles, cellulolytic bacteria need to attach to the surface. Under low pH values, the growth and binding ability of many cellulolytic bacteria is impaired (Russell et al., 2009). Low intracellular pH values restrict bacterial growth and fermentation processes. Therefore, the pH value affects cellulolytic degradation of feed material by restricting enzymatic activity and inhibiting growth and binding capacity of the bacteria (Russell and Wilson, 1996, Russell et al., 2009). Roger et al. (1990) reported the largest number of adherent cells for the cellulolytic Fibrobacter succinogenes between pH 5.5 and 7.0, concluding that enzymatic inducted binding activity of F. succinogenes is greatest in those pH ranges. Furthermore, the
pH may also affect the electrostatic interactions between surfaces. However, not all cellulolytic bacteria are unable to attach in low pH values. There are many different mechanisms of attachment and complex enzymes like cellulosomes of anaerobic bacteria and fungi (Aurilia et al., 2000) affecting cellulose degradation (Schülein, 2000).
Contrarily to F. succinogenes, the adhesion of Ruminococcus flavefaciens and R.
albus is not impaired by lowered pH (Rasmussen et al., 1989, Roger et al., 1990).
Nevertheless, cellulolytic activity can still be observed below a ruminal pH 6.0, however, at a much reduced rate (Hiltner and Dehority, 1983). Cellulolytic bacteria can provide cellodextrins to more acid resistant noncellulolytic bacteria (Russell and Dombrowski, 1980b, Mouriño et al., 2001). Studies imply that cellulolytic bacteria do not lose the ability to digest cellulose below pH 6.0, however, growth rate is highly impaired (Slyter, 1986, Mouriño et al., 2001). The ruminal pH needs to remain above pH 6.0 to support bacterial growth in order to maintain cellulose digestion. Terminating pH values are below 5.3 (Mouriño et al., 2001). The ability to digest cellulose below pH 6.0 is of some significance, as rumen pH values in modern dairy and beef production remain in these pH ranges for a significant amount of time during feeding cycles (Russell et al., 2009). Nevertheless, some anaerobic microorganisms are able to grow in acidotic environments (Weimer, 1992). They are able to create a low pH gradient by decreasing the intracellular pH value accordingly to the outer pH reduction. This prevents from SCFA accumulation. The bacterial strategy to reduce the intracellular pH can only be beneficial, if enzymes are tolerant to low pH values and this does not impair the intracellular metabolism. One of these pH resistant bacteria is Streptococcus bovis. In low pH environments, the intracellular pH decreases, however, acetate, formate and ethanol are then no longer produced by S. bovis. Instead, the fermentation
pattern shifts to homolactic production, indicating that intracellular enzymes are affected by low pH and determine the fermentation outcome (Russell and Hino, 1985).
In vivo, the adaptation to an intense concentrate feeding scheme, resulting in decreased ruminal pH values, leads to a shift in the microbial population. After an increase of lactic acid-producing bacteria, the abundance of lactate utilizing bacteria increases as the rumen adapts to high concentrate rations (Tajima et al., 2000).
Generally during SARA, the ruminal lactate concentration does not exceed 5 mM (Owens et al., 1998b). Sun et al. (2010) increased the concentrate ratio over a period of 45 d and reported a gradual change in fermentation patterns, microbial diversity and abundance. Authors reported a proliferation of associated bacteria according to the increasing concentrate ratio and a general increase of propionate during the trial. In their study, butyrate producing bacteria proliferated until they disappeared when 50%
concentrate was fed, which was also reflected in the fermentation pattern. When feeding a 70% concentrate ratio most fibrolytic-related species disappeared, while starch fermenting and acid tolerant bacteria like S. bovis and Prevotella spp. persisted.
The decrease of high abundant and diverse cellulolytic species during the shift from high-forage to high-concentrate rations is also reported in several other in vivo studies (Weimer et al., 1999, Yang et al., 2001). Generally, the phyla Firmicutes and Bacteroidetes are reported to be the main phyla within the rumen (Petri et al., 2013).
During rumen acidosis, observations regarding the bacterial alterations vary. Several studies report an increase of the relative abundance of the phylum Firmicutes, when SARA was induced by excessive grain feed (Fernando et al., 2010, Mao et al., 2013, Plaizier et al., 2017). Mao et al. (2013) concluded that the increase of this phylum is due to an increase in bacterial species, which are able to cope with an elevated
availability of carbohydrates. Contrarily, Watanabe et al. (2019) reported a decreasing abundance of Firmicutes during acidosis, while Bacteroidetes tended to increase in low pH values. In contrast, in other studies Bacteroidetes is often found to diminish during low rumen pH values, as most bacterial species belonging to Bacteroidetes are Gram-negative and are sensitive to low pH values (Khafipour et al., 2009c, Huo et al., 2014). The discrepancy in the bacterial distribution may result from individual reaction to SARA induction (Mohammed et al., 2012) and different alterations within the main phyla, due to different feed rations (Duarte et al., 2017, Arik et al., 2019). The phylum Fibrobacteres and the family Ruminococaceae are predominantly cellulolytic and their abundance is enhanced in fiber rich diets (Roger et al., 1990). During concentrate rich feeding schemes with low rumen pH values, the abundance of Bacteroidetes is diminished, while Bifidobacteriaceae and certain species of Prevotellaceae are increased (Mao et al., 2013). Therefore, it is desirable to aim for a high phylogenetic resolution when analyzing the bacterial diversity within the rumen.
1.4 In vivo models for subacute rumen acidosis
Subacute rumen acidosis does not only affect the fermentation pattern, but has also a substantial impact on animal welfare (Abdela, 2016) and economic consequences for the farmer. Therefore, rumen acidosis has been a major topic of scientific research in the past years and will continuously be studied as knowledge within the field progresses. Many in vivo studies have been performed in the past decades. However, the protocol for SARA induction varies between studies. In several studies, the basic feed is replaced by a sudden increase of concentrate in the ration (Keunen et al., 2002, Krause and Oetzel, 2005, Vyas et al., 2015). To ensure a proper intake of the SARA-
ration, the animals are often restrictively fed the day before SARA induction. Most studies use wheat or barley pellets, or a mixture of both to reduce the ruminal pH below 5.6 (Krause and Oetzel, 2005), below 6.0 and 5.6 (Keunen et al., 2002), or below 5.8 (Vyas et al., 2015), respectively. Furthermore, it is possible to force feed animals within a certain time, creating a mean pH of 5.59, as performed in the study of Schlau et al.
(2012). A third option is to use ruminally cannulated animals and to apply a high amount of grains directly into the rumen (Lettat et al., 2010, Kmicikewycz and Heinrichs, 2014). These studies report a mean SARA pH of 4.85 to 6.09, and pH 5.72 to 5.51, respectively. However, recently, most studies induce SARA by simply increasing the concentrate proportion within the daily ration (Metzler-Zebeli et al., 2013, Plaizier et al., 2017, Arik et al., 2019), resulting in pH values below pH 5.6.
To measure the ruminal pH development, especially indwelling 24 h-pH sensors are commonly used in in vivo studies with non-cannulated cows (Nocek et al., 2002).
Furthermore, besides a permanently cannulation of animals, there are currently two methods available for ruminal sampling in vivo. Firstly, an oral stomach tube, and secondly rumenocentesis providing access to the rumen. In an in vivo study, Duffield et al. (2004) compared both methods to a 24 h-continuous rumen pH measurement with indwelling pH sensors in ruminal cannulated cows. The study implied that results differ among sampling procedures. The most sensitive technique was the rumenocentesis method. The oral stomach tube revealed higher pH values and higher bicarbonate concentrations, compared to samples collected by rumenocentesis.
However, great expense and ancillary costs for in vivo studies limit these experiments to a small number of animals. A great advantage of fistulated animals is the precise localization of the sampling, which cannot be accomplished by an oral stomach tube
(Weimer, 2015). Mohammed et al. (2012) implied that the individual response to SARA induction protocols varies significantly among animals, which means that a greater number of animals would be statistically desirable. Furthermore, the impact on animal welfare needs to be considered. To overcome these problems, in vitro models have been established to observe rumen fermentation and microbial diversity.
1.5 In vitro models for subacute rumen acidosis
The analysis of rumen fermentation is rather difficult in vivo. Therefore, several in vitro models have been developed to observe the rumen fermentation process in controlled laboratory surroundings, aiming to mimic in vivo conditions. The in vitro models can be divided in two major groups, batch culture systems (Hoover et al., 1976) and continuous culture models (Czerkawski and Breckenridge, 1977). The systems may be modified, depending on the aim of the study. Isolated microorganisms can be observed (Chen et al., 2019) as well as complex microbial interactions (Strobel et al., 2007). Batch culture fermentation systems are the most frequently used in vitro methods to observe ruminal fermentation processes (Broudiscou and Lassalas, 2000) and specific microbial interactions (Vries et al., 1970, Chen et al., 2019). Anele et al.
(2015) evaluated models for acidosis prediction using batch culture models. By diluting the buffer solution, pH decreased below 5.2. The batch culture model allows a large number of samples within one run, and benefits from a space saving setup. Using batch culture systems, liquid rumen content and buffer solutions are incubated in gas tight glass flasks. Fermentation gasses escape into attached syringes. The temperature is maintained by a heating system and the pH is regulated by applying HCl and NaOH solutions. After a certain time a lack of substrate and the accumulation
of microbial waste products limit the growth and activity rate (France and Dijkstra, 2005).
Using continuous flow models, the negative effects of the unchanged milieu is encountered by a continuous removal of waste products and a steady supply of substrates. This enhances the establishment of a steady state within the microbial community and the model can be used for several days (Busquet et al., 2005) extended to weeks (Fraser et al., 2007). Continuous flow models reflect the in vivo situation closer compared to batch culture approaches (Stern et al., 1997). In these models, rumen liquid and solid content is continuously diluted with a buffer solution. The effluent is either pumped out or allowed to overflow (France and Dijkstra, 2005). The pH is maintained by using HCl and NaOH solutions (Mansfield et al., 1995, Fuentes et al., 2009). Several studies use a modified dual-flow system (Calsamiglia et al., 2002, Cerrato-Sánchez et al., 2007), which was originally established by Hoover (1976).
Briefly, in these set ups, flow rates of the buffer solution, and the liquid and solid components can be adjusted. The solid and liquid components are allowed to stratify within the vessels, providing different liquid and solid flow rates (Teather and Sauer, 1988). Generally, the duration of these studies vary from hours (Long et al., 2014) to days (Lourenço et al., 2008).
This thesis presents an in vitro experimental trail using the rumen simulation technique (Rusitec), established by Czerkawski and Breckenridge (1977). The Rusitec provides a single-flow semi-continuous culture approach. Contrarily to the dual-flow, the buffer solution and the effluent flow are continuous, and the pH is not regulated by HCl and NaOH, but via buffer dilution and modifications. Furthermore, the feed ration is provided only once a day. The design of the model is described later in the text (3.2
Experimental set up: The Rumen Simulation Technique). One of the first SARA inductions in a Rusitec model was described by Oliveira et al. (1996), who observed the microbial metabolism of thiamine regarding the development of polioencephalomalacia in cattle. In this study, SARA was induced by lowering the amount of buffer salts in the buffer solution, resulting in a pH decrease between 5.17 and 5.79 pH units. Recently, Rusitec studies have been performed by Eger et al. (2017) and Mickdam et al. (2016), observing the impact on microbial populations during rumen acidosis. In the study of Eger et al. (2017), a severe rumen acidosis was induced by lowering the buffering substances within the artificial saliva, resulting in pH values below 5.0 pH units and a decrease in the microbial community. After the acidosis challenge, a recovery period was induced by infusing the standard buffer solution and authors reported a regeneration of the microbial population and fermentation products.
Mickdam et al. (2016) induced SARA (pH 5.6–5.7) in a Rusitec model and evaluated microbial changes during acidosis by applying a PCR analysis. However, the analyses did not include a regeneration period following the acidosis challenge.
In this thesis, we used the Rusitec model to induce a subacute rumen acidosis under laboratory conditions. Firstly, we aimed to observe the changes in fermentation patterns during acidosis and, innovatively, after acidotic conditions during the regeneration period. In a second step, the bacterial population was analyzed, by combining two next generation sequencing approaches. The goal was to observe the changes within the ruminal community during SARA and, furthermore, to monitor the ability of the bacterial community to recover from acidotic challenges in the Rusitec system.
2 Material and methods 2.1 Animals
Two ruminally fistulated, non-lactating Holstein cows were used in this experimental set up to collect fresh ruminal inoculum for each experimental run. The previous fistulation was approved by the Lower Saxony State Office for Consumer Protection and Food Safety (LAVES) by the experiment number AZ 33.4-42505-04-13A373. The animals were kept on straw and fed 7.5 kg of hay, and 500 g of a rye and wheat based concentrate. Solid and liquid inoculum was collected 3 h after feeding.
2.2 Experimental set up: The Rumen Simulation Technique
The rumen simulation technique (Rusitec) is one of the most commonly used semi continuous culture systems for rumen fermentation (Mateos et al., 2017a), originally established by Czerkawski and Breckenridge (1977). The Rusitec model consists of fermentation vessels in which ruminal fermentation processes can be monitored over a long period of time. Most Rusitec based studies last several days or even weeks (Mateos et al., 2017a). The Rusitec intends to simulate the rumen function and therefore, provides compartments for two fractions of microbes – liquid associated microorganisms, and solid associated microorganisms. However, a compartment for the epimural associated microorganisms cannot be provided by the Rusitec model.
This experimental trial was performed using two Rusitec models. The first Rusitec consists of six identical fermentation vessels (a) made of polymethylmethacrylate with a volume of approximately 750 ml. The second only contained two vessels, which were contained with a continuous pH measurement device. In both devices, the vessels were placed in a water bath (b) in which a thermostat (Thermomixer®, Braun
Melsungen AG, 34209 Melsungen) constantly maintained a temperature of 39°C, to mimic body temperature. The vessels consisted of an outer vessel and an inner, movable vessel. The outer reaction vessel was filled with fresh liquid rumen content at the start of each experiment. This ruminal fraction contained the liquid associated microorganisms (LAM). The inner vessel contained two nylon bags (c) (16 cm × 7 cm, porous size 150 µm, Linker KG, 34127 Kassel), which provided the feed ration. At the beginning of the trial, one nylon bag was filled with solid rumen content to transfer the solid associated microorganisms into the Rusitec vessel. On the following day, the nylon bag containing the rumen content was replaced with a new feed bag.
Henceforward, every day feed bags were exchanged alternatingly, ensuring a retention time for each bag of 48 h. To transfer the microbial fraction, before replacement, the feed bag was flushed with pre-warmed buffer solution for 1 min. The solution was then poured back into the open fermentation vessel. The lid and the bottom of the inner vessel were perforated. To ensure the best transfer from nutrients and microorganisms into both fractions, an electric motor (d) moved the inner vessel up and down (6 rpm) to mimic rumen motility. A pump (e) (Typ B1, Ole Dich, Hvidore, Dänemark) constantly infused buffer solution (f) into the fermentation vessel via a Tygon® tube (4.0 mm × 7.2 mm, Omnilab GmbH & Co. KG, 30989 Gehrden), ensuring a daily liquid turnover rate of 100%. The composition of the buffer solution was close to the components of the ruminant’s saliva and therefore maintained a physiological pH value in the fermentation vessels. The effluent and gasses left the vessel through a butyl tube (8.0 mm × 12.0 mm, YMC Europe GmbH, 46539 Dinslaken). The effluent was continuously collected in glass bottles (g), which were stored in Styrofoam boxes (h) and cooled on ice for further sampling. Gasses escaped the system through siphons
on top of the effluent bottles. To maintain the anaerobe environment, the effluent flasks were gassed with nitrogen after each time the system has been opened.
Figure 1 The Rusitec model: this figure shows a diagrammatic model of the Rumen Simulation Technique applied in this experimental trial
2.3 Experimental design and sampling scheme
Each experiment consisted of an equilibration period of 7 days, a first control period (5 days, CP I), the acidosis period (5 days, AP) and ended after the regeneration period (control period II;; CP II) of 5 days.
During the equilibration period, the pH and redox potential were measured once per day, when the reaction vessels were opened for the daily feed bag exchange. During CP I, AP, and CP II fluid samples were collected from the effluent bottles on a daily basis and stored at -20°C until further treatment. The effluent samples were used to analyze lactate, SCFA and ammonia-N concentrations. To monitor the degradation process, feedbags were collected once in each period. On these sampling days, the concentrate ration and the hay ration were applied in two separate bags. Therefore, both could be dried and weighted and the individual degradation rate could be calculated. On the last day of each period, additional samples of the fluid and solid phase were collected to analyze the liquid (LAM) and solid associated microbes (SAM).
Figure 2 Experimental design and sampling schedule
2.4 Measurement of fermentation parameters 2.4.1 pH and redox potential
The pH and the redox potential were measured once daily, after opening of the fermentation vessel, with a pH meter (Digital-pH-Meter 646, Knick GmbH & Co. KG, 14163 Berlin) and pH and redox sensors (Polyplast pH Sensors and Polyplast ORP Sensors, Hamilton Bonaduz AG, Switzerland). The redox potential was measured for 60 s. The pH sensor was calibrated every 3 d with a standardized solution of pH 4.01 and pH 7.00 (Mettler-Toledo GmbH, 35353 Gießen). Two of the eight fermentation vessels were equipped with a continuous computerized measuring device. For the complete experimental run of 22 d, the pH and redox potential was measured every 10 s. The average was protocoled and recorded every 5 min using a data logging program.
2.4.2 Lactate
Samples for lactate concentration were collected from daily effluent, which was individually measured for each fermentation vessel. The concentration of D- and L-
lactate was measured using a commercial kit (Milchsäure D-Laktat/ L-Laktat;;
Boehringer Mannheim/ R-Biopharm, Enzymatische BioAnalytik / Lebensmittelanalytik;;
Roche, Mannheim, Germany). Preparatory, the samples were centrifuged at room temperature for 10 min at 25830 g. Then, 1 ml of the supernatant was transferred into an Eppendorf-tube. The pH of the sample material was adjusted to pH 8 – 10 using 1 M NaOH. The samples were kept at -20°C until analysis. After defrosting, the samples were centrifuged for 10 min at 13000 g until they were treated as recommended by the manufacturer’s instructions. The determination of lactate concentration is based on the
