Lab 7 Ruminal fluid Flashcards
Indications for ruminal fluid examination
- Diagnosis of ruminal disease
- Evaluation of ruminal fluid before use in therapeutic transfusion (transfaunation)
How to obtain ruminal fluid?
- Orogastric tube
- Ruminal puncture
Orogastric tube method
- For obtaining ruminal fluid
- Cow is restrained
- Stomach tube put down the nasal passage or through the mouth, measure tube beforehand to be sure it reaches rumen, tube smaller than 1,5 cm may be obstructed
- Speculum fixes mouth to prevent chewing of tube -< tube into esophagus -> tube then swallowed, confirmed in rumen by auscultation simoultaneously with air blown into tube -> distinctive gas smell will come out
- Stirrup pump attached to tube withdraw the sample
- First portion discarded, containing a lot of saliva -> elevates pH of fluid
- Be aware of trachea, kink the tube when pulling out
Ruminal puncture method
- For obtaining ruminal fluid
- In sterile conditions, left side of animal, inserting a needle in ventral rumen, aspirating fluid sample, 3-5 ml
- Site is marked: horizontal line level with top of patella about 15-20cm posterior to last rib
- Not proper sample for all type of examinations
Transportation of ruminal fluid sample?
For long distance: in double jacket container
Estimation of chloride and ammonia concentration of rumen fluid sample?
Can be delayed up to 9hours in room temp. and up to 24hours in refrigerator
Factors influencing the ruminal fluid composition, how to minimize exposure to environment?
- Minimize effects of cooling and air exposure on protozoal activity
- Physical examinations from fresh sample
- Samples should be taken min. 4-6hours from feeding, and animal should not drink before sampling
Factors influencing the ruminal fluid composition
- Composition of the diet
- Circumstances of the feeding and sampling
- Time interval from feeding or drinking
Samples should be taken min. 4-6hours from feeding, and animal should not drink before sampling. Why?
- Fermentation will be more active immediately after feeding and therefore VFA and gas conc. increase, whilst pH will be lower.
- Drinking water will dilute and may decrease temp of sample, influence conc. of different substances, decrease the motility of microorganisms, and decrease viscosity of sample
Physical examination of ruminal fluid; how, where
Should be performed on the site of animal, as physical characters of the ruminal fluid will be rapidly chancged after collection
Physical examination of ruminal fluid; Odor
Normal: aromatic odor
Abnormal:
- ammonia smell (urea posioning)
- moldy rotting (protein putrefaction)
- acidic or sour odor (excess lactic acid/grain overfeeding)
Physical examination of ruminal fluid; Color
Normal:
- olive to brownish green (hay rotation)
- deeper green color (green rotation)
- yellowish brown color (grain or silage rotation)
Abnormal:
- milky grey (grain overfeeding, lactic acidosis)
- darker greenish or brownish (ruminal stasis/decomposition)
- grey with clots of milk (calves with abomasal reflux)
Physical examination of ruminal fluid; Consistency
Normal: slightly viscous
Abnormal:
- increased viscosity (saliva contamination)
- decreased viscosity, watery with few feed particles (inactive bacteria or protozoa)
- excess frothy, stable bubbles (frothy bloat/vagus indigestion)
Physical examination of ruminal fluid; Sedimentation time or sedimentation/flotation test
Allow fluid to sit in a test tube and determine the time for complete sedimentation and flotation of solid particles. Smaller particles sink, larger particles float on the bubbles of fermentation.
- Normal range: 4-8min
- Very active fluid: may exhibit sedimentation of fine particles with subsequent flotation
- Inactive fluid: shows rapid sedimentation with little to no flotation, due to lack of fermentative gases
- Rumen acidosis, prolonged anorexia, indigestible feeds with inactive flora
- Stable froth presence indicates frothy bloat or some types of vagal indigestions in the Hoflund disease (stenosis, hypermotility) or in case with treatment with sympatiomethics (acetylcholine) used for the treatment of ruminal atonia
- May be no sedimentation or flotation
Biochemical examination: pH measurement
- Best measured 2-4 hours after feeding concentrates or 4-8 hours after feeding a total mixed ratio.
- It can be measured with pH paper or portable pH meter paper
- Samples collected by rumenocentesis have lower pH values than those collected by oral routes
- Rumenocentesis samples were about 0.3 pH units lower than rumen fluid collected simultaneoulsy through rumen canal
Biochemical examination: normal pH
- Range between 6.3-7 (slightly acidic)
- Cows fed grain can have slightly lower, those fed hay or green slightly higher pH
Biochemical examination: abnormal pH, elevated pH
Rumen alkalosis
- Simple indigestion or reduced feed intake for more than 2 days
- Urea indigestion
- Putrefaction of ruminal content from prolonged rumen stasis
- Saliva contamination
Biochemical examination: abnormal pH, lowered pH
Rumen acidosis
- Grain overfeeding
- Chronic ruminal acidosis
Note:
- an animal with lactic acidosis may have a normal rumen fluid pH if anorexia has been prolonged due to continued saliva production
- abomasal reflux generally results in little to no change in rumen pH, making rumen chloride a better choice for elevation
VFA: principle VFAs
Acetic, propionic and butyric acids
VFA: ratio and pH ratio
Varying with diet, but majority is acetate
pH: average ratio pH 6-7 is 60-65% acetic acid, 20-25% of propionic acid, 10-15mol% butyric acid and 5mol% other acids
- In normal conditions lactic acid is not present in rumen fluid
- If crude fiber content is around 20% the acetic acid:propionic acid ratio is 4:1
VFA: decrease in VFA content and effect on pH
- A decrease in VFA content indicates a diet low in fibers or sufficient intake, and decreased activity of the microbial organisms
- High grain diet decrease pH of ruminal fluid
VFA: how to measure it
VFA can be measured with high performance liquid gas chromatography (HPLC). The concentration of VFA in the rumen is between 80-120 mmol/L. It is the highest around 3 to 5 hours after feeding
VFA: When pH is between 5-6:
Ratio of acetic acid decreases (<40%), whilst propionic acid (25-30mol%) and butyrate (20-30mol%) increase
VFA: If pH is lower than 5.4-5.6:
The lactic acid (up to 10mol%) increase, and the other vFA decrease (propionic acid, butyrate)
VFA: In case of feeding diet low in fiber, the acetic acid:propionic acid ratio will be
Narrow until 1 or 14
Concentration of VFA in rumen
The concentration of VFA in the rumen is between 80-120 mmol/L. It is the highest around 3 to 5 hours after feeding
Tests for the reducing ability of the anaerobic rumen flora
- Redox potential (oxidation-reduction) is constant in the rumen and is dependent on the activity of anerobe microorganisms in the rumen
- Mostly bacteria have important role in the redox processes in the rumen. Their dehydrogenating acitivty can be shown with different color reagent. Some of them will be reduced to colorless forms (methylene blue)
- Reduction is done mostly by the organisms which play a role in carbohydrate fermentation
Mostly bacteria have important role in the redox processes in the rumen. Their dehydrogenating acitivity can be shown with different color reagent: methylene blue
Reduced to colorless forms
Mostly bacteria have important role in the redox processes in the rumen. Their dehydrogenating acitivity can be shown with different color reagent: Triphenyltetrazolium-chloride,TTC test
From colorless oxidized form to reduced colored form
Tests for the reducing ability of the anaerobic rumen flora; reduction is performed mainly by?
- Reduction is done mostly by the organisms which play a role in carbohydrate fermentation
Tests for the reducing ability of the anaerobic rumen flora; what reduces and increases the redox potential?
- In diet rich in starch during ruminal acidosis -> redox potential increased
- When the ruminal flora is destroyed (ex. putrefaction) -> redox potential reduced
Nitrite reduction test;
Test the capability of the ruminal fluids bacteria to reduce the nitrites and the nitrates
Nitrite reduction test; procedure
- Potassium nitrate (KNO2) to ruminal fluid in 2 test tubes -> warm water baths
- Put to ceramic plate and add one drop Griess-Ilosvay reagent
- In presence of nitrite: color pink
- Normally no nitrite after 10min in healthy ruminal fluid, because microorganisms are reducing the nitrite
- Reduction is more rapid when cattle are fed green fodder or have ruminal decomposition or bloat, and slower when a deficient ration is fed or inapetence
Methylene blue reduction test;
Tests for reducing ability of the anaerobic rumen flora
Methylene blue reduction test; procedure
- Mix fresh rumen fluid and methylene blue in test tube
- Let it stand and determine time taken to decolorize sample until only a blue ring remains at the top of sample
Methylene blue reduction test; interpretation
Normal: 3-6 min, concentrate diets results in faster times, forage diets result in longer time of range
Prolonged: discoloration which takes longer tham 10-15min indicates inadequate anaerobic bacterial population, rumen acidosis or indigestible roughage
The TTC test is performed in the same way as? In this case we expect there result to be?
- Same as Methylene blue reduction test
- Expect red coloration after incubation. From the colorless TTC in 10-15min we have red colored product (formazan) by the metabolic activity of bacteria
Ruminal flora and fauna;
The microbes in the rumen include bacteria, protozoa and fungi. Bacteria and protozoa are the predominant microbes.
Ruminal flora and fauna; what are the most predominant microbes and how much to they digest
- Bacteria and protozoa are the predominant microbes
- Digest about 70-80% of the digestible dry matter in the rumen
Ruminal flora and fauna; Protozoa
- Both ciliates and flagellates are present in the rumen, but only ciliates are of physiological importance by virtue of their number and mass
- Sensitive to ruminal environment
- Not essential for forestomach digestion, but capable of many tasks
Ruminal flora and fauna; how many species to one animal normally contain?
About 10-20 different species
Ruminal flora and fauna; living conditions of ruminal ciliates
Strict anaerobic conditions
Ruminal flora and fauna; Protozoa very sensitive to changes in ruminal environment and their inactivity will show
Earlier derangement of the ruminal homeostasis
Ruminal flora and fauna; Protozoa are not essential for forestomach digestion, but they have many tasks?
- Breaking down soluble sugars and various polysaccharides or storing them (starch, hemicellulose, xylane, pectin)
- They are capable to prevent rapid fall in ruminal pH by utilizing some starch that otherwise would be broken down by bacteria
- Their active movement help at mixing ruminal content
Ruminal flora and fauna; microscope examination
- One drop of fresh rumen fluid on a slide and observe unstained
- See groups of protozoa: small, medium and large. Should also be rapidly moving
- Number and motility is judged as follows: highly motile and very crowded (+++/+++), sluggish motility and low numbers (+/+), no alive protozoa (0/0)
Number and motility of Protozoa are judged in a microscope slide:
highly motile and very crowded (+++/+++)
sluggish motility and low numbers (+/+)
no alive protozoa (0/0)
Ruminal flora and fauna; motility of ciliates and protozoa
- In healthy ruminal fluids they are moving very fast, with the same intensity
- Motility is reduced both in acidosis and alkalosis
- Motility of protozoa is reduced when the environmental changes are induced rapidly
- In case of chronic processes, they do not disappear completely, but their number and motility is reduced
- Always the biggest sized protozoa will be affected first
Ruminal flora and fauna; motility of ciliates and protozoa, Errors?
- If the temp of the ruminal fluid is decreased, the slide is cold
- If temp is decreased, motility of protozoa will be reduced and fluid will be judged as +++/0
Ruminal flora and fauna; bacteria
- Vital for fermentation processes of forestomach
- Conc. varies between 10^7 and 10^12 per ml of fluid rumen
- Fewer bacteria is present when the ration is rich in cellulose (crude fiber) compared with ratio rich in starch
- ## Categorized in functional groups
Ruminal flora and fauna; bacteria categorized in functional groups
- Fibrolytic types
- Amylolytic types
- Proteolytic types
Ruminal flora and fauna; amolytic and proteolytic bacterial flora comprises species
Streptococcus, lactobacillus
Ruminal flora and fauna; fibrolytic bacterial flora comprises species
Streptococcus, sarcina, spirillacea and selenomonas
Ruminal flora and fauna; how to examine bacteria
Staining or culturing
- Staining: Giemsa, Gram or PAS
Ruminal flora and fauna; Gram staining of bacteria reveals?
Populations of bacteria present
Ruminal flora and fauna; normal rumen fluid contains mainly what type of bacteria
Gram negative bacteria
Ruminal flora and fauna; normal rumen fluid contains mainly gram-negative bacteria, but what can alter the populations
- Lactic acidosis: results in a more homogenous, predominantly gram-positive population
- Diet rich in fiber: gram-negatives are predominant
- Diet rich in starch: gram-positives are in higher conc.
Ruminal flora and fauna; culturing bacteria
- Usually requires anaerobic conditions
- In case of ruminal putrefaction, Proteus, Clostridium and Pseudomonas are present in high conc.
Fungi
- Represented mostly by yeasts, ex. candida spp
- May contribute to carbohydrate, mostly cellulose, digestion
Fungi; can be revealed by?
Giemsa, PAS staining or culturing
Fungi; what happens in case of acidosis or putrefaction?
Theu disappear completely from the rumen, or they multiple on the place of bacteria
Rumen fluid ammonia;
- Ammonia is synthesized from protein and non-protein nitrogen (NPN) sources by the ruminal bacteria
- It is absorbed through the ruminal wall and passes further through the portal vein
- Ammonia is used as energy source or is built up in the bacterial protein
Rumen fluid ammonia; measurement
- Spectrophotometer or with ion selective method
- Difficult because NH3 is highly volatile, and environmental NH3 (stable, smoke) will contaminate the sample
- Sample should be immediately measured, or transported in closed system between 0-2C
Rumen fluid ammonia; interpretation of measurement
- Normal level: low, 6-20mmol
- Ammonia will increase in case of high dietary protein intake or in energy deficiency. Increased NH3 will lead to ruminal alkalosis and in severe cases to ammonia intoxication
- Liver disorders also can cause increased ammonia levels. After calving the liver function is reduced because of energy deficiency and fatty infiltration
Increased NH3 will lead to?
Ruminal alkalosis and in severe cases to ammonia intoxication
Rumen fluid chloride, normal range
Chloride conc. is low in healthy animals, 15-20mmol/L
Rumen fluid chloride, main source
Diet and saliva which passes into the rumen
Rumen fluid chloride, what can increase the conc.?
Gastric torsion or other types of pylorus obstruction will make the hydrogen chloride pass into the rumen /reflux phenomenon) and can increase chloride up to 30-100mmol/L
Rumen fluid chloride, measurement
- Measured in a supernatant of a centrifuged sample
- Relatively stable over time: can be stored at room temp (9hours) and refrigerated (24hours) and still be accurate
- Spectrophotometry method
Rumen fluid chloride, interpretation of measurement
- Normal level: <30 mEq/L (15-20 mEq/L) and in sheep <15 mEq/L
- Elevated level: high salt intake, abomasal diplacement, obstruction of intestinal flow (ileus, pyloric stenosis, abomasal disorders (inflammation, ulcer, hyperacidity)
