Lab 7 - Ruminal Fluid

Question 1

List the methods we use to obtwin ruminal fluids and what the ruminal fluids indicate

Answer 1

orogastric tube and ruminal puncture

• Diagnosis of ruminal diseases
• Evaluation of ruminal fluid before use in therapeutic transfusion (transfaunation).

Question 2

obtain ruminal fluid By orogastric tube: what do we need to do in preparation

Answer 2

Before:

1. Measure length of tube required so it ends in rumen
2. Diameter more than 1.5cm, ingesta can cause obstruction
3. Dental gag is inserted to protect tube from teeth

Question 3

obtain ruminal fluid By orogastric tube: method

Answer 3

Method

1. Cow is restrained
2. Stomach tube through nasal passage or through dental gag in mouth, into the esophagus
3. The tube is passed down during swallowing
4. We know the tube is in the rumen when air comes out ithbits distinct odor of fermented gas
5. A pump is used to exract the sample
6. The initial sample is discarded bc high in saliva which elevates the pH
7. The tube is kinked and removed in a downward motion to prevent loss of ruminal fluid and so it doesnt get into the trachea

Question 4

Describe ruminal puncture: method

Answer 4

• in sterile conditions on L side of animal
• not suitable for all examinations!
  1. Insert needle into venteal rumen at level of prox end of patella, 15-20com caudal from last rib

Question 5

Transport and handling of ruminal fluid sample

Answer 5

-Transportation of ruminal fluid for long distance must be done in double jacket container. -Estimation of chloride and ammonia conc. can be delayed up to 9 hrs in room temperature and up to 24 hrs in refrigerator

Question 6

Factors influencing the ruminal fluid composition

Answer 6

• cooling and air on protozoal activity
• composition of the diet
• the circumstances of the feeding and sampling
• the time interval from feeding, Min 4-6h after feeding - Fermentation is more active after eating which incr VFA and gas conc - decr. pH
• animal should not drink before sampling: dilute sample, decr temp, decr motility of microorganisms, decr viscosity.

Question 7

Physical examination of ruminal fluid - when

Answer 7

By side of animal, fresh sample

Odor, color and consistancy

Question 8

Odor

Answer 8

Normal:
􏰁 Aromatic odor

Abnormal:
􏰁 Ammonia smell (urea poisoning)
􏰁 Moldy rotting (protein putrefaction)
􏰁 Acidic or sour odor (excess lactic acid/grain overfeeding)

Question 9

Color

Answer 9

Normal:
􏰁 Olive to brownish green (hay ration)
􏰁 Deeper green color (green ration)
􏰁 Yellowish brown color (grain or silage ration)

Abnormal:
􏰁 Milky grey (grain overfeeding, lactic acidosis)
􏰁 Darker greenish or brownish (ruminal stasis/decomposition)
􏰁 Grey with clots of milk (calves with abomasal reflux)

Question 10

Consistency

Answer 10

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)

Question 11

Sedimentation time or sedimentation/flotation test

Answer 11

Very active flora - may be sedimentation - flotation

Inactive flora - rapid sedimentstion- no flotation

Question 12

Elevated pH (Rumen alkalosis)

Answer 12

STASIS! Goes together with anorexia as not enough food, energy goes to maintaining pH causing putrefaction!

1️⃣Simple indigestion or reduced feed intake for greater than 2 days - both decr VFA
2️⃣Too much NH3 from protein synth so cannot be transformed to urea. Carbs are needed to digest proteins so NH3 accumulation)

3️⃣Putrefaction of ruminal content from prolonged rumen stasis (consequence not a cause!)
4️⃣ Saliva contamination(pH 8.5) false positive, wrong sampling!

Question 13

􏰁 Lowered pH 5.5.-6 (Rumen acidosis)

Answer 13

1️⃣Acute acidosis: Grain overfeeding, quickly digested to VFA causing acute acidosis!
2️⃣Chronic ruminal acidosis. Acute leads to chromic by overfeeding starch. The bacteria will remodel (lactobacillus) (decr eating, tired)

Question 14

VFA can be measured with

Answer 14

high performance liquid gas chromatography (HPLC), highest around 3-5h after feeding

Question 15

reducing ability of the anaerobic rumen flora is dep on and can be measured how

Answer 15

Redox potential (oxidation-reduction) is constant in the rumen and is dependent on the activity of anaerobe microorganisms in the rumen. Their dehydrogenating activity can be shown with different color reagent.

Question 16

List the tests used for the red ability of the anaerobic rumen flora and what they do

Answer 16

Nitrite reduction test
Test the capability of the ruminal bacteria’s to reduce the nitrites and nitrates.

Methylene blue reduction test
Tests for the reducing ability of the anaerobic rumen flora.

The TTC test, same as meth bluein result and procedure

Question 17

Method of Nitrite reduction test

Answer 17

• KNO2 solution is added to ruminal fluid in test tubes. Then they are heated for different durations and then a drop of griess-Ilosvay reagent is added

Pink - nitrite is present. After 10mins there should be none since the flora reduce the nitrite (normal-colorless!!)

Decreased red ability during ruminal alkalosis! Decr feeding, incr feeding etc

Question 18

Method of methylene blue test

Answer 18

Methylene blue and ruminal fluid is mixed in a test tube. Measure the time it takes for de-colorization until only blue ring on top of sample remains in normal samples.

􏰀 Normal:
3–6 minutes; concentrate diets result in faster times, forage diets result in longer time of range
􏰀 Prolonged:
Discoloration which takes longer than 10–15 minutes indicates inadequate anaerobic bacterial population, rumen acidosis or indigestible roughage

Question 19

TTC test

Answer 19

The TTC test (triphenyltetrazolium-chloride) is performed in the same way. In this case we expect a red coloration after incubation. From the colorless TTC in 10-15 minutes we have a red colored product (formazan) by the metabolic activity of bacteria. Normal is color

Question 20

What is Ruminal flora and fauna

Answer 20

The microbes in the rumen include bacteria, protozoa and fungi. Bacteria and protozoa are the predominant microbes. They digest about 70% to 80% of the digestible dry matter in the rumen.

Question 21

Protozoa morphology and environment conditions

Answer 21

• ciliates(important) and flagellates(not important)
• anaerobic conditions
• sensitive to changes in ruminal environment and their inactivity will show earlier the derangement of the ruminal homeostasis

Question 22

Protozoa functions

Answer 22

• breaking down soluble sugars and various polysaccharides (starch, hemicellulose, xylane, pectin) or storing them
• they are capable to prevent rapid fall in ruminal pH by utilizing some starch that otherwise would be broken down by bacteria (to VFA)
• their active movement help at the mixing of the ruminal content

Question 23

Microscopic examination of protozoa

Answer 23

• one drop of fresh rumen fluid on a HEATED microscope slide
• observe UNSTAINED under 160-200 X power.
• Should see three groups of protozoa: small, medium and large and they should be rapidly moving.

very crowded and Highly motile
+++/+++

low numbers and Sluggish motility +/+

No alive protozoa 0/0

Question 24

What influencs the motility and number of protozoa

Answer 24

Motility ⬇️: alkalosis, acidosis, oxygen, chronic processes

Number⬇️: During chronic processes

The biggest are always affected first

Errors: if the temperature of the ruminal fluid is decreased, the slide is cold. If the temperature is decreased, the motility of the protozoa will be reduced, and the fluid will be judged as +++/0.

Question 25

Bacteria function

Answer 25

vital for the fermentation processes of the forestomach

Question 26

Bacterias present in rumen

Answer 26

Usually gram-, but i if the lactic acidosis the gram+ dominates, and much startch gram- dominates

There are more bacteria present if more starch (than cellulose)

Gram staining can be used to examine bacteria! PAS and giemsa too. Culture is hard since as strickly anaerobic

Question 27

Fungi

Answer 27

• represented mostly by yeasts
• may contribute to carbohydrate (mostly cellulose) digestion
• Giemsa, PAS staining or culturing.
• acidosis or putrefaction: they disappear completely from the rumen, or they multiple on the place of bacteria.

Question 28

Rumen fluid ammonia: production, measurement

Answer 28

(Ruminal alkalosis)

• synthesized from protein and non-protein nitrogen (NPN) sources by the ruminal bacteria - absobed through ruminal wall
• spectrophotometer or with ion selective method - since its volatile and nh3 in air makes it more difficult.

Question 29

interpretation of Rumen fluid ammonia:

Answer 29

• Normal level in rumen is low
• more protein, more hn3 -> ruminal alkalosis, 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.

Question 30

Rumen fluid chloride production, measurement

Answer 30

• from diet and the saliva
• measured in a supernatant of a centrifuged sample. spectrophotometer or ion selective method. 9h in room temp, 24 in fridge

Question 31

Interpretation of Rumen fluid chloride production

Answer 31

Normal level is low
Elevated level:
1️⃣ High salt intake
2️⃣ Abomasal displacement or Obstruction of intestinal flow the hydrogen chloride will pass into the rumen (reflux phenomenon) and can increase chloride up to 30-100 mmol/l.
3️⃣ abomasal disorders (inflammation, ulcer, hyperacidity)

Question 32

Normal pH of rumen

Answer 32

6.3-7 (slightly acidic)