Exam 2 Flashcards
Is rumen bacteria large?
No, its small! 10^10-10^11 cells/mL ruminal fluid
What is the replication time of rumen bacteria?
Generally short replication time. Most 20-60 min (can be 48+ hrs)
Cellulolytics are highest on which diet?
Highest number on a high forage diet. Lower numbers on a high starch diet (but still present)
What are some prominant Cellulolytic species?
- Ruminococcus albus
- Ruminococcus flavifaciens
- Fibrobacter succinogenes
- Butyrivibrio fibrisolvens
What are Cellulolytics responsible for in the rumen?
- primary H producers in the rumen
- symbiosis with methanogens, other H utilizers
What do Cellulolytics produce?
Cellulase a membrane-bound enzyme complex of several subsequent enzymes (cellulosome)
How does pH effect cellulolytics?
Very sensitive to pH change fiber digestion begins to decrease below pH 6.0 growth severely retarded below 5.5
Where doe cellulolytics get their Nitrogen from?
- Ammonia (NH3)
- very limited use of amino acids, peptides
Growth of cellulolytics is stimulated by what?
Branched-chain VFA isobutyrate, isovalerate, 2-methylbutyrate
What are hemicellulolytics or pectinolytics?
- simple sugar degraders
- Sucrose, maltose, cellobiose, dextrins Increase with high grain, lush pasture
- Most species of bacteria
What is the reaction that takes place with hemicellulolytics/pectinolytics?
- HC/Pectin –> Cellobiose + pentoses –> glucose Pentoses released for use by other species
Amylolytics increase on what diet?
- Increased #’s on a high starch diet lower #’s but always present on a high forage diet
- Some cellulolytics capable of starch digestion
What are some important amylolytic species?
- streptococcus bovis
- prevotella ruminocola
- bacteriodes amylophylus
What do amylolytics produce?
Amylase extracellular enzyme activity regulated by pH
What do amylolytics such as S. bovis directly produce?
Lactic acid (lactate) etiology of lactic acidosis
Where do amylolytics find their source of Nitrogen?
- NH3 and amino acids+peptides
- Max growth requires AA+ peptides
- Require 2/3 of N supply from AA+peptides provision of pre-formed protein vs. NPN
What do intermediate acid utilizers use?
Lactate, succinate, and other organic acids
What are some important intermediate acid utilizer species?
- Megasphaera elsdenii
- Selenomonas ruminantium
What do intermediate acid utilizers play an important role in?
- adaptation to high grain diets
- S bovis produces lactate excess lactate –> lactic acidosis
- M. elsdenii converts lactate to propionate
What do proteolytics produce?
- Major NH3 producers in the rumen protein –> peptides –> AA –> NH3 NH3 production greatly exceeds own reqts.
- Use C-skeleton of AA for energy
What are some important proteolytic species?
- Peptostreptococcus anerobius
- Clostridium stricklandii
- NH3 production is 10x higher than mixed rumen microbes identification via monesin sensitivity (Strickland)
Bacteria use what as an energy source?
Simple sugars no cellulase or amylase rely upon CHO digestion by other species
T/F: Methanogens are a true bacteria
False, they are archae
T/F: Methanogens need oxygen to survive
False, they are strict anaerobes
fWhat do methanogens use from cellulolytics?
Require and use Hydrogen CO2+ 4H2 –> CH4 +2 H2O
What pH are methanogens sensitive to?
Low pH
Lower #’s on a high grain diet (pH 5.5-6.0)
What is methanogen growth inhibited by?
- monensin methanogens are NOT overtly monensin sensitive
- Restricted supply of H
- less H produce fewer (fewer Gram +)
- Increased utilization of other H sinks
Acetogens are direct competitors with what?
- methanogens compete for Hydrogen
- lower affinity for H than methanogens
- methanogens outcompete acetogens for H manipulate acetogens to reduce methanogenesis
Ureolytics produce what?
Urease Urea–> CO2 +2 NH3 Membrane-bound enzyme
Where is there a higher population of ureolytics?
- Rumen wall
- play a role in recycling of blood urea to rumen
- maintain low intra-ruminal urea concentrations ensures positive concentration gradient for diffuesion
Lipolytics produce what?
Lipase extracellular and membrane-bound forms
What do lipolytics ferment?
- Glycerold is fermented propionate (energy)
- Fatty acids are not fermented in the rumen
- Biohydrogenation
Biohydrogenation of unsaturated fatty acid by Gram + bacteria is what kind of mechanism?
Detoxification mechanism conformation of cis double bonds in SFA vs. USFA
Biohydrogenation of USFA by Gram + bacteria plays what role in the body?
- Role in FA composition of stored body fat
- Conjugated Linoleic Acid
- Rumenic acid
- anticarcinogenic (breast cancer)
_________________ have fewer numbers than bacteria, but up to equal mass. (larger than bacteria)
Protozoa
What percentage of microbial Nitrogen in the rumen do Protozoa produce?
20-50% remain resident in rumen attachment to larger particles, rumen wall contribute to intestine N outflow is negligible
What are the two general classes of protozoa
- Holotrichs
- Endtrodiniomorphs
Describe Holotrich Protozoa.
- Low #’s
- Large Diameter
- Long replication time (38 h+)
- Very sensitive to low pH (<6.0)
- Exist mostly on high forage diets
Describe Entodiniomorphs
- Highest #’s
- smaller diameter
- most have shorter replication time (~8hrs)
- Tolerant of low pH (can survive <5.5 for short time)
- Survive well on high grain diets
Protozoa digest only____________________.
Nonstructural
CHO do NOT digest fiber (no cellulase) engulph larger feed particles produce high levels of propionate & butyrate
Protozoa____________ starch.
- sequester/store
- role in moderating lactic acidosis keeps starch away from amylolytic bacteria
Protozoa often engulph too much ___________.
Starch Overload–> cell lysis
Protozoa have ___________ #’s with high __________ diets.
- Generally lower #’s with high grain diets no holotrichs (pH too low)
- Fewer entodiniomorphs
Protozoa does not use what as a Nitrogen source?
NH3 require pre-formed protein (AA, peptides) have proteolytic capability can make significant contribution to NH3 pool
Protozoa are called miniruminants because___?
predation of other microbes engulph bacteria (or smaller protozoa) sometimes called miniruminants cellulolytics survive inside protozoa
Though fungi are large, most are smaller than _______
protozoa
Fungi have ______ numbers in the rumen and ______ turnover from rumen. They replicate via ______ formation.
Low numbers in the rumen low turnover from rumen replicate via spore formation
Fungi digest ______ using ____________. ______ aid in breaking apart strands of fiber which expose cellulose to bacteria.
- Fungi digest cellulose using fungal cellulase.
- Hyphae aid in breaking apart strands of fiber to expose cellulose to bacteria.
Explain why fungi is low in #’s despite role in fiber digestion.
hard to establish, maintain large population inoculation increases fiber digestibility chronic inoculation required ongoing research focus fungal extracts (cellulase)
What are the three types of interactions?
- parasitism (one benefits, other harmed)
- commensalism (one benefits, no effect on other)
- mutualism (both benefit)
- -often increases efficiency of both species
- -removal of feedback inhibition
Utilization of intermediates/endproducts include
Succinate, lactate, ethanol, formate, H2, CO2 accumulate in pure culture not detected (or very low) in mixed culture
When crossfeeding with proteolytics, what are the products of protein breakdown?
- NH3 AA and peptides
- C-skeletons of AA
- -BCVFA (from BCAA- Val, Ile, Leu)
- -Aromatic compounds (from aromatic AA- Phe, Tyr)
When crossfeeding with proteolytics, what is utilized by other species?
- NH3 used by cellulolytics, most other species
- -synthesis of AA BCVFA, aromatic compounds used by cellulolytics
- -synthesis of corresponding BCAA, aromatic AA
Protozoal interactions will likely include…
- Engulphing large particles in rumen -bacteria (attached to feed particles “Mini-ruminants”
- -cellulolytic bacteria digest cellulose for protozoa
- -protozoa digest bacteria
- – use bacteria as a source of AA
- –supply NH3, AA/Peptides, BCVFA, etc to other species
Nutrient limitations account for what?
- The variation in microbial #’s
- -normal eating patterns
- – max populations 2-6 h after feeding
- -Rate of CHO fermentation
- -normal eating patterns
_____________ are highly susceptible to nutrient limitations
- Intermediate acid utilizers
- Selenomonas ruminantium (ST50= 2.5 h)
- Megaspheara elsdenii (ST50= 3-5h)
A shift in the fermentation pathway causes?
- A higher ATP yield
- -more acetate, less propionate
- -can be between or within species
Microbes are efficient scavengers of____?
NH3 low saturation at cell wall
What are two methods of Nitrogen incorporation into the cell?
- Glutamine Synthetase
- Glutamate Dehydrogenase
Describe Glutamin Synthetase.
- High affinity for NH3
- One mole ATP per mole of NH3 fixed
- Spend energy incorporating N when NH3 is limiting (high affinity, works at low NH3, requires ATP)
Describe Glutamate dehydrogenase
- Low affinity for NH3
- No ATP requirement
- Majority of NH3 fixation at higher NH3 (Low affinity, needs high NH3, no ATP required)
If there is a change in diet, microbes adapt how?
- most all microbes present at all times (shift in proportion only)
- Transition time depends on magnitude of change
If you go from a forage diet to a high concentrate diet, what occurs?
- Increased rate of fermentation
- -increased VFA concentration = lower pH
- Selection against pH sensitive species
- -decreased cellulolytics, increased amylolytics
- decreased methanogens
- general decrease in protozoa
- -fewer below 6.0
- –entodiniomorphs can withstand short-term pH below 5.5
- -role in moderating acidosis
- engulf starch –> less lactate
- engulf too much starch –> lysis
- -fewer below 6.0
What is lactic acidosis?
- accumulation of lactate in rumen
- insuficient IAU population to utilize lactate
- lactate is stronger acid (pH may drop < 5.0)
- low ruminal pH
- inhibits ruminal contractions
- damage to rumen epithelium –> liver abscesses
- low pH inhibits amylase activity in rumen
- scours
- high starch content (gray appearance)
- intestinal casts (repair of damage to intestinal wall)
- wide fluctuation in feed intake
- off feed –> hunger –> off feed –> hunger
How do you avoid lactic acidosis?
- Need to increase IAU population
- S Bovis (20 min) vs. M elsdenii (48 h)
- Gradual step up program is required
- 3-4 wk adaptation protocol
- add s10-20% grain to diet every 3-4 days
- allows time for increase in population of M. eldenii to match the rate of lactate production
- concerns once adapted
- feeding mistakes, weather change, processing
- may need to add long forage to diet and readapt
What are some ionophore antibiotics (VFD exempt)?
Monensin (Rumensin) <– Coban (for poultry)
Lasalosid (Bovatec)
Laidlomycin (Cattlyst)
What is ionophores mechanism of action?
- ion transport channels in cell membrane
- increases ion flux across cell membrane
- microbe enteres futile ion cycle
- available energy expended pumping ions (Na pump)
- increased maintenance energy req.
- less energy available for growth
Ionophores inhibit growth of Gram + bacteria
Ionophores may depress ______ digestion
Fiber digestion (short term)
major cellulolytics are Gram +
Ionophores have a major effect on __________.
Methanogens
Secondary effect of less H2
- decreased acetate: propionate ration
- increased capture of energy as VFA
- improved feed efficiency
- selects agains major NH3 producers
- increased peptides, AA
How do ionophores influence protozoa?
protozoa are monensin sensitive (coccidiostat)
Originally used in poultry (coban)
How do ionophores influence feed intake?
Decreased total intake, increased eating frequency
smaller meals –> decreased acidosis
streptococcus bovis
Amylolytic bacteria
selenomonas ruminantium
Intermediate acid-utilizing bacteria
ruminococcus flavifaciens
cellulolytic bacteria
peptostreptococcus anaerobius
proteolytic bacteria
ophryoscolex caudatus
entodiniomorph protozoa
methanobacterium ruminantium
methanogen
megaspheara elsennii
intermediate acid-utilizing bacteria
isotricha intestinalis
Holotrich protozoa
fibrobacter succinogenes
cellulolytic bacteria
entodinium caudatum
entodiniomorph protozoa
dasytricha ruminantium
Holotrich protozoa
clostridium stricklandii
proteolytic bacteria
butyrivibrio fibrisolvens
cellulolytic bacteria
bacteroides amylophylis
amylolytic bacteria
anaerovibrio lipolytica
lipolytic bacteria
Do ruminal protozoa have the ability to digest cellulose? Explain your answer.
No, they do not have cellulase. However, since they often engulf cellulolytic bacteria who then live inside them like mini-ruminants, they do have some cellulose digestion inside them because of the cellulolytic bacteria
’s relative to other protozoa (high vs. low)
Entodiniomorph vs. Holotrich
size relative to other protozoa (large vs. small)
sensitivity to low pH (tolerant vs. tolerant)
typical replication time (in hrs.)
Entodiniomorph:
- # ’s relative to other protozoa: high
- size relative to other protozoa: small
- sensitivity to low pH: tolerant
- typical replication time: 8 hrs
Holotrich
- # ’s relative to other protozoa: low
- size relative to other protozoa: large
- sensitivity to low pH: sensitive
- typical replication time: 36 hrs
List and compare two primary enzyme systems used by rumen bacteria for fixing nitrogen. For each enzyme, indicate 1) the optimum relative NH3 concentration required for activity of each enzyme and 2) energy use in the reactions catalyzed by these enzymes.
Microbes us glutamine synthetase and glutamine dehydrogenase to fix NH3. GD requires a higher concentration and microbes prefer to use it because it does require any ATP or cost them any energy. They use GS when NH3 concentrations are low/limited, but it does require ATP.
What influences does short-term starvation have on bacterial numbers in the rume. Which bacteria are most susceptible to such nutrient limitations, and why? What changes in cellular metabolism do these microbes make in response to nutrient limitation and how does this influence VFA production in the rumen?
Short term starvation decreases the overall number of microbes in the rumen. Intermediate acid utilizers are most susceptible, because they have a long replication time which makes it hard to build the population back up when some die. To compensate, microbes shift their ferementation pathways to get a higher ATP yield and they produce more acetate than proprionate.
Moorella thermoacetica
Acetogen
Describe the primary structural difference between gram positive and gram negative bacteria. Which type of bacteria are susceptible to monensin, and how does monensin affect the growth of these susceptible bacteria.
Gram positive bacteria are susceptible to monensin. This is due to only having 2 layers for the monensin to penetrate into the cell and effect ion flow (only one membrane). Monensin uses an ion flow mechanism to kill the bacteria. Using the Na/K pump, monensin pushes more K into the cell forcing the pump to use more ATP to pump Na out of the cell to maintain ion concnetration. This results in lower Na, higher pH and lower substrate utilization inside the cell and lower K and lower ATP inside the cell. This is a futile cycle that leaves too little ATP for the bacteria to reporduce.
What role do acetogens play in the rumen? What substrates do these microbes utilize, and what endproduct do they produce? What is the underlying reason why there are few acetogens in the rumen, and why migh higher numbers of these microbes in the rumen be beneficial?
Acetogens utilize H to produce acetate. They have a lower affinity for H than methanogens which is why their numbers are low. They would be more beneficial because they produce acetate rather than methane, which is more efficient.
What specific location within the rumen would the majority of ureolytic bacteria be found and why? What enzymes do these microbes produce and what is the role of these microbes in the process of recycling blood urea into the rumen?
Ureolytics are found in the rumen wall because they have closer access to the blood. They take in urea from the blood and bring it into the rumen. Their enzyme urease turns urea into CO2+ NH3, which can be used by other microbes.
What effect do ionophors have on both intake patterns and overall feed intake in cattle? How does this effect relate to use of ionophores as a tool to moderate ruminal acidosis and why?
Ionophores decrease feed intake. The presence of monensin triggers the satiety center of the brain, telling them they are full. The cattle eat smaller, more frequent meals, rather than overeating in one sitting. This decreases acidosis because there isn’t such a large influx of starch.
As a nutritoinist for the feedlot in the previous question, it is alos your duty to train employees of the feedlot on how to recognize and prevent ruminal acidosis. Begin your training program by describing the outward signs/symptoms of acidosis that the employees should be looking for and when and how these assessments should be conducted. In addition, discuss what feeding practices should be followed to prevent acidosis from occuring and your approach to correcting a situation in which a significant number of calves in a pen are exhibiting signs of acidosis.
- Symptoms
- grey feces
- this is caused by the increased starch levels going through the intestine. The starch is femented in the hind gut and increases VFA’s and other products in the large intestine. This increases fecal water content, so the feces will be loose.
- Going on/off feed
- cattle with acidosis will gorge then go off feed, then gorge again. This occurs because their belly hurts due to a low pH in the rumen which is burning the rumen wall. They feel better once the rumen is empty and are really hungry by that point, so they overeat and the cycle begins again.
- Mucous cast
- once over acidosis , the cattle will pass a mucous cast. This is the mucous coating that lines the small intestine. It served as a protective barrier against lactic acid, but damage will cause it to pass and rebuild.
- grey feces
- Employees should
- check daily for symptoms because acidosis will drastically decrease gains and can lead to death
- be sure to feed multiple smaller feedings throughout the day to avoid overeating
- watch closely as the weather changes. cattle will gorge before storms, typically
- be wary of feeding mistakes. Changes will affect the cattle!
- If acidosis occurs
- offer longstem forage to calves to decrease amylolytic populations, increase pH and improve the health of rumen microbe populations
Describe the importance of branched-chain fatty acids to the rumen microbe population. Include in your answer 1) the three most common BCFA 2) how these BCFA are formed and by which class of ruminal microbes and 3) which other class of ruminal microbes requires BCFA for optimal growth and for what purpose
Proteolytics produce BCVFA’s by deamination of AA and BCAA. Cellulolytics use BCVFAs to synthesize AAs and BCAA. They reaminate BCVFA to AA and BCAA. This provides AA and BCVFA for utilization by other microbes. The 3 BCVFA are isobuterate, 2 methylbuterate, and isovalerate.
Describe the process of biohydrogenation by ruminal microbes. Include in your answer which ruminal microbes perform this biohydrogenation and the underlying reason why they do so.
The rumen microbes prefer SFA to incorperate into their cell membrane for structural intergrity. Gram positive bacteria perform biohydrogenation.
Why would it be beneficial to maintain a viable population of protozoa in the rumen of a steer fed a high grain diet? Include in your answer at least two reasons why maintaining that population of protozoa can be difficult when high grain diets are fed.
Protozoa digest starch which would be used by amylolytics. By protozoa using it, less lactic acid is produced. Maintaining the population can be hard because holotrichs have slow replication and are sensitive to pH. Also, protozoa are less likely to over eat starch and go through lysis.
Descibe the life cycle (replication process) employed by ruminal fungi. Include in your answer a discussion of how this process influences overall cellulose digestion within the rumen.
Fungi are spore replicators. Zoospores embed in plant material and begin to grow. They root into the plant particle, using a rhizoid. This then branches out which helps break up the plance material, exposing more cellulose. Fungal cellulase is excreated, allowing the fungi to utlilize the cellulose. The fungus grows and reproduces and the cycle continues. Although fungi numbers are relatively low in the rume, they greatly influence cellulose digestion. The breaking up of the plant material exposes more cellulose for cellulolytics to digest.
