RUMINAL FLUID

EXAMINATIONS

Dr. Eva A Ajaj

Msc. Clinical pathology

Indication of Ruminal fluid examination

-Diagnosis of ruminal diseases

-Evaluation of ruminal fluid before use in

therapeutic transfusion

Methods of collection:

-Needle puncture of the rumen

-Oral or nasal passage of stomach tube

General precaution for rumen fluid examination:

-Samples should be evaluated directly after

collection to minimize effects of cooling and air

exposure on protozoal activity.

-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 temp. and up to

24 hrs. in refrigerator.

Examination of ruminal fluid

Physical characters:

-Color

-Consistency

-Odor

-Sedimentation activity test

Chemical characters:

-pH

-Cellulose digestion test

-Glucose fermentation test

-Nitrate reduction test

-Rumen fluid chloride

Microscopical exam:

-Quantitative exam.

-Qualitative exam

Physical characters

-Color

Normal : Olive to brownish green (hay

ration) Deeper green color (green ration)

Yellowish brown color (grain or silage

ration)

Abnormal: Milky grey (grain overfeeding)

Darker greenish (ruminal

stasis/decomposition) Grey with clots of

milk (calves with abomasal reflux)

Physical characters

-Consistency

Normal: Slightly viscous

Abnormal: Watery ( Inactive bacteria or protozoa)

Excess frothy (Frothy bloat/ vagus indigestion)

-Odor

Normal: Aromatic odor

Abnormal: Ammonia smell (Urea poisoning) Moldy

rotting (protein putrefaction) Sour odor (excess lactic

acid/grain overfeeding)

Chemical Examination:

pH: The pH may be measured by using indicator paper having

a specificity of 0.2 pH (Fig 7-2), or, better, an electrical pH

meter. Normal pH ranges between 6-7 in animal on a mostly

forage diet, but is lower 5.5-6.5 in animals fed mostly grain.

Elevated pH (Rumen alkalosis):

o Simple indigestion.

o Urea indigestion.

o Putrefaction of rumen ingestion.

o Feeding on indigestible roughage.

Lowered pH (Rumen acidosis)

o Chronic rumen acidosis (pH 5-5.5).

o Abomasal reflex from abomasal disease.

o Vagal indigestion.

o Intestinal obstruction.

Sedimentation activity test (Evaluation of microfloral activity)

This test provides a rapid evaluation of microfloral activity.

Put a sample of rumen fluid in test tube and let to stand.

Measure the time needed for completion of sedimentation,

which is referred to as the sediment activity time.

Normal time is 4-8 minutes.

Abnormal time may be:

o Very rapid sedimentation with no floatation occurs in rumen

acidosis, prolonged anorexia, inactive microflora from

indigestible roughage.

o No appreciable sedimentation and floatation in frothy bloat,

some cases of vagal indigestion

Methylene blue reduction test

This test reflects the anaerobic fermentation metabolism of

bacterial population.

Mix 20 ml of rumen content with 1 ml of 0.03% methylene

blue in a test tube and let to stand at room temperature.

Measure the time needed for color of the mixture to be

changed.

Normal rumen fluid from cattle fed on a hay and grain diet

needs 3 minutes to decolorize leaving a narrow ring of blue color

at the top of decolorizing mixture.

Abnormal reduction of time up to 15 minutes indicates

indigestible roughage, anorexia of several days, or rumen

acidosis

Cellulose digestion test:

Mix 10 ml of rumen fluid with 0.3 ml of 16% glucose solution in a

test tube.

Immerse a thread of pure cellulose (free from synthetic fiber).

The lower end is weighted by a glass bead (Fig 7-5).

Incubate the tube at 39˚c.

Record the time for the bead to be dropped free at the bottom of

the tube.

A fully active rumen fluid will digest the cellulose within 48-56

hours.

The test takes a long time and is not very accurate.

-Glucose fermentation test 0.5 ml of 16% glucose + 10 ml. of rumen fluid

Place the mixture in a fermentation saccharometer

Keep the saccharometerat 39˚C

Read the results after 30 –60 min.

The test measure indirectly the ability of ruminal flora to ferment

glucose through measuring the volume of formed gas

Normal microflora (1-2 ml gas production / 1hour)

Inactive microflra (little or no gas formation)

Nitrate reduction test

It is provide an idea on activity of microbes that synthesize nitrogen compounds.

10 ml of sieved ruminal fluid is placed into each of 3 test tubes and 0.2, 0.5, 0.7 ml of

0.025% potassium nitrate solution is added to 3 tubes.

Put the 3 tubes in water both at 39˚c. Every 5 minutes, one drop from each tube is placed in

the small ceramic plate.

To each drop is added 2 drops of reagent 1 and 2 drops of reagent2.

Observe the change of color.

Sample that contain nitrates are colored red.

Rumen fluid of cattle fed a mixed ration will not change in color after 5-10 minutes in tube

1 and 20 minutes in tube II, and 30 minutes in tube III.

Reduction is more rapid when cattle are green fodder or have ruminal decomposition or

bloat.

Reduction is more slower when a deficient ration is fed or when the animal lacks the

appetite

1 Reagent I: 2 ml of sulphanilic acid in 30% acetic acid to make 200 ml.

2 Reagent II: 0.6 ml alpha-naphthylamine + 16 ml concentration acetic acid + 140 ml D.W

Rumen fluid chloride

-Measured in a supernatant of a centrifuged sample

-Measured by chloride meter.

-Normal level is : 30 mg/l

-Elevated level :

Abomasal disease.

* Abomasal reflux.

* Obstruction of intestinal flow

Microscopical examination

Qualitative method -Prepare a fresh film.

-Examine by low power.

-The activity of the fauna is judged

as follow:

Motility Activity

-Highly motile and very crowded +++

-Motile and crowded ++

-Sluggish motility and low numbers +

-No or sporadic alive fauna 0

2. Quantitative evaluation of rumen fluid fauna.

Technique

-Strained rumen fluid sample.

-Dilute 1 ml of strained sample with 15 ml saline solution and 5 ml lugol`s iodine

solution and shake gently.

- Spread 0.1 ml of the mixture on glass slide in an area under cover glass of 22 X 50

mm.

- Counting is carried out using low power (X 10). The field area of that lens is one

square millimeter mm2 .

- Count 30 fields in the slide. The average count in 30 fields represents the protozoal

count over one square millimeter area of the field(30*10).

- Multiply the average by 1100(22*50) to have protozoal count in 0.1 mm of the diluted fluid

which represents 0.02(1ml/50) ml of the original sample.

- Multiply the obtained figure by 50(1ml/0.02) to obtain total protozoal count per ml.

50*1100*N/30= protozoal count/mm3

Examination of the Bacteria

Bacteria of the fore stomachs are vital for ruminants.Their concentration

varies between 107and 1012/ml of rumen fluid contents or /gram of solid

contents. Standard bacteriological techniques such as direct counting and

culture for identification, differentiation and colony counts have so far

found no application in the clinical examination of rumen fluid.

Microscopical observation of the fore stomach microflora is useful for

diagnosis of rumen acidosis. Air-dried smear of rumen fluid is stained by

Gram's method, or other staining methods as nigrosine, Congo red,

modification of Giemsa stain can be applied to fresh or fixed specimens

Criteria used for interpretation of the smear are:

Presence or absence of morphologically distinguishable bacterial species

characteristic of a normal rumen flora, which called "leading bacteria".

The multiplicity or uniformity forms.

The ratio of Gram-positive to Gram-negative bacteria.

A diet rich in starch produces a more uniform picture, with

Gram-negative cocci, short and long rods, and a relatively

high proportion of Gram-positive cocci and rods

A digestive disorder involving ruminal inactivity or

decomposition may be suspected when the leading bacteria

associated with a particular ration are absent, when there is

unusual uniformity in the microflora, or when there are

bacteria that are not normally present

Gram stained smears from rumen fluid samples can be prepared. There

are mainly Gram negative bacteria in normal rumen fluid, but in

ruminal acidosis Gram positive streptococci and lactobacilli

predominate

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