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Plain tubes (i.e. without any anticoagulant) are used for chemistry studies after separation of serum: liver function tests (total proteins, albumin, aspartate aminotransferase, alanine aminotransferase, bilirubin), renal function tests (blood urea nitrogen, creatinine), calcium, lipid profile, electrolytes, hormones, and serum osmolality. Fluoride bulb is used for collection of whole blood for estimation of blood glucose. Addition of sodium fluoride (2.5 mg/ml of blood) maintains stable glucose level by inhibiting glycolysis. Sodium fluoride is commonly used along with an anticoagulant such as potassium oxalate or EDTA.
The International Council for Standardization in Haematology (ICSH) was initiated as a standardization committee by the European Society of Haematology (ESH) in 1963 and officially constituted by the International Society of Hematology (ISH) and the ESH in Stockholm in 1964. The ICSH is recognised as a Non-Governmental Organisation with official relations to the World Health Organisation (WHO).
 
The ICSH is a not-for-profit organisation that aims to achieve reliable and reproducible results in laboratory analysis in the field of diagnostic haematology.
 
The ICSH coordinates Working Groups of experts to examine laboratory methods and instruments for haematological analyses, to deliberate on issues of standardization and to stimulate and coordinate scientific work as necessary towards the development of international standardization materials and guidelines.
Anticoagulants used for hematological investigations are ethylene diamine tetra-acetic acid (EDTA), heparin, double oxalate, and trisodium citrate (Table 791.1).
 
Table 791.1 Salient features of three main anticoagulants used in the hematology laboratory
Salient features of three main anticoagulants used in the hematology laboratory
 
Ethylene Diamine Tetra-acetic Acid (EDTA)
 
Changes occurring due to prolonged storage of blood in EDTAThis is also called as Sequestrene or Versene. This is the recommended anticoagulant for routine hematological investigations. However, it cannot be used for coagulation studies. Disodium and dipotassium salts of EDTA are in common use. International Committee for Standardization in Hematology recommends dipotassium EDTA since it is more soluble. It is used in a concentration of 1.5 mg/ml of blood. Dried form of anticoagulant is used as it avoids dilution of sample. Its mechanism of action is chelation of calcium. Proportion of anticoagulant to blood should be maintained. EDTA in excess of 2mg/ml causes shrinkage of and degenerative changes in red and white blood cells, decrease in hematocrit, and increase in mean corpuscular hemoglobin concentration. Excess EDTA also causess welling and fragmentation of platelets, which leads to erroneously high platelet counts. Prolonged storage of blood in EDTA anticoagulant leads to alterations as shown in Figure 791.1 and Box 791.1. EDTA is used for estimation of hemoglobin, hematocrit, cell counts, making blood films, sickling test, reticulocyte count, and hemoglobin electrophoresis.
 
Preparation
 
Dipotassium EDTA 20 gm
Distilled water 200 ml
 
Mix to dissolve. Place 0.04 ml of this solution in a bottle for 2.5 ml of blood. Anticoagulant should be dried on a warm bench or in an incubator at 37°C before use. For routine hematological investigations, 2-3 ml of EDTA blood is required.
 
Changes in blood cell morphology crenation of red cells separation of nuclear lobes of neutrophil vacuoles in cytoplasm and irregular lobulation of monocyte and lymphocyte nuclei due to storage of blood in EDTA anti
Figure 791.1 Changes in blood cell morphology (crenation of red cells, separation of nuclear lobes of neutrophil, vacuoles in cytoplasm, and irregular lobulation of monocyte and lymphocyte nuclei) due to storage of blood in EDTA anticoagulant for prolonged time
 
Heparin
 
Heparin prevents coagulation by enhancing the activity of anti-thrombin III (AT III). AT III inhibits thrombin and some other coagulation factors. It is used in the proportion of 15-20 IU/ ml of blood. Sodium, lithium, or ammonium salt of heparin is used. Heparin should not be used for total leukocyte count (since it causes leukocyte clumping) and for making of blood films (since it imparts a blue background). It is used for osmotic fragility test (since it does not alter the size of cells) and for immunophenotyping.
 
Double Oxalate (Wintrobe Mixture)
 
This consists of ammonium oxalate and potassium oxalate in 3:2 proportion. This combination is used to balance the swelling of red cells caused by ammonium oxalate and shrinkage caused by potassium oxalate. Mechanism of anticoagulant action is removal of calcium. It is used for routine hematological tests and for estimation of erythrocyte sedimentation rate by Wintrobe method. As it causes crenation of red cells and morphologic alteration in white blood cells, it cannot be used for making of blood films.
 
Preparation
 
Ammonium oxalate 1.2 gm
Potassium oxalate 0.8 gm
Distilled water upto 100 ml
 
Place 0.5 ml of this solution in a bottle for 5 ml of blood. Anticoagulant should be dried in an incubator at 37°C or on a warm bench before use.
 
Trisodium Citrate (3.2%)
 
This is the anticoagulant of choice for coagulation studies and for estimation of erythrocyte sedimentation rate by Westergren method.
 
Preparation
 
Trisodium citrate 3.2 gm
Distilled water upto 100 ml
 
Mix well to dissolve. Store in a refrigerator at 2-8°C.
 
Use 1:9 (anticoagulant: blood) proportion for coagulation studies; for ESR, 1:4 proportion is recommended.
 
ESR should be measured within 4 hours of collection of blood, while coagulation studies should be performed within 2 hours.
 
Further Reading:
 

ABO Grouping

There are two methods for ABO grouping:

  • Cell grouping (forward grouping): Red cells are tested for the presence of A and B antigens employing known specific anti-A and anti-B (and sometimes anti-A, B) sera.
  • Serum grouping (reverse grouping): Serum is tested for the presence of anti-A and anti-B antibodies by employing known group A and group B reagent red cells.

Both cell and serum grouping should be done since each test acts as a check on the other.

There are three methods for blood grouping: slide, tube and microplate. Tube and microplate methods are better and are employed in blood banks.

Further Reading:

  1. Autoagglutination: Presence of IgM autoantibodies reactive at room temperature in patient’s serum can lead to autoagglutination. If autocontrol is not used, blood group in such a case will be wrongly typed as AB. Therefore, for correct result, if autocontrol is also showing agglutination, cell grouping should be repeated after washing red cells with warm saline, and serum grouping should be repeated at 37°C.
  2. Rouleaux formation: Rouleux formation refers to red cells adhering to each other like a stack of coins and can be mistaken for agglutination. Rouleaux formation is caused by high levels of fibrinogen, immunoglobulins, or intravenous administration of a plasma expander such as dextran. Rouleaux formation (but not agglutination) can be dispersed by addition of normal saline during serum grouping.
  3. False-negative result due to inactivated antisera: For preservation of potency of antisera, they should be kept stored at 4°-6°C. If kept at room temperature for long, antisera are inactivated and will give false-negative result.
  4. Age: Infants start producing ABO antibodies by 3-6 months of age and serum grouping done before this age will yield false-negative result. Elderly individuals also have low antibody levels.
D antigen is the most immunogenic after ABO antigens and therefore red cells are routinely tested for D. Individuals are called as Rh-positive or Rh-negative depending on presence or absence of D antigen on their red cells. Following transfusion of Rhpositive blood to Rh-negative persons, 70% of them will develop anti Rh-D antibodies. This is of particular importance in women of childbearing age as anti-D antibodies can crosss the placenta during pregnancy and destroy Dpositive fetal red cells and cause hemolytic disease of newborn. In other sensitized individuals, reexposure to D antigen can cause hemolytic transfusion reaction.
 
In Rh D grouping, patient’s red cells are mixed with anti-D reagent. Serum or reverse grouping is not carried out because most Rhnegative persons do not have anti-D antibodies; anti-D develops in Rh-negative individuals only following exposure to Rh-positive red cells.
 
Rh typing is done at the same time as ABO grouping. Method of Rh D grouping is similar in principle to ABO grouping. Since serum or reverse grouping is not possible, each sample is tested in duplicate. Dosage effect (stronger antigenantibody reaction in homozygous cells i.e. stronger reaction with DD) is observed with antigens of the Rh system. Autocontrol (patient’s red cell + patient’s serum) and positive and negative controls are included in every test run. Monoclonal IgM anti-D antiserum should be used for cell grouping, which allows Rh grouping to be caried out at the same time as ABO grouping at room temperature. With monoclonal antisera, most weak and variant forms of D antigen are detected and further testing for weak forms of D antigen (Du) is not required. Differences between ABO and Rh grouping are shown in Table 788.1.
 
Table 788.1 Comparison of ABO grouping and Rh typing
Comparison of ABO grouping and Rh typing
Microplate is a polystyrene plate consisting of 96 micro wells of either U- or V-shape. Grouping is carried out in micro wells. This method is sensitive and ideal for large number of samples (see Figure 787.1).
 
Further reading: Rh D GROUPING METHOD
Principle
 
Red cells from the specimen are reacted with reagent antisera (anti-A and anti-B). Agglutination of red cells indicates presence of corresponding antigen (agglutinogen) on red cells.
 
Specimen
 
Capillary blood from finger prick, or venous blood collected in EDTA anticoagulant.
 
Reagents
 
ABO antisera: See box 786.1 and Figure 786.1.
 
BOX ABO antisera
Box 786.1: ABO antisera
 
Anti A and anti B sera used for cell grouping
 Figure 786.1 Anti-A and anti-B sera used for cell grouping
 
Method
 
  1. A clean and dry glass slide is divided into two sections with a glass marking pencil. The sections are labeled as anti-A and anti-B to identify the antisera (see Figure 786.2).
  2. Place one drop of anti-A serum and one drop of anti-B serum in the center of the corresponding section of the slide. Antiserum must be taken first to ensure that no reagents are missed.
  3. Add one drop of blood sample to be tested to each drop of antiserum.
  4. Mix antiserum and blood by using a separate stick or a separate corner of a slide for each section over an area about 1 inch in diameter.
  5. By tilting the slide backwards and forwards, examine for agglutination after exactly two minutes.
  6. Result:
    Positive (+): Little clumps of red cells are seen floating in a clear liquid.
    Negative (–): Red cells are floating homogeneously in a uniform suspension.
  7. Interpretation: Interpret the result as shown in the Table 786.1 and Figure 786.2.
 
Table 786.1 Interpretation of cell grouping (forward grouping) by slide test
Anti-A Anti-B Blood Group
+ - A
- + B
+ + AB
- - O
 
Cell grouping by slide method
Figure 786.2 Cell grouping by slide method
 
Slide test is quick and needs only simple equipment. It can be used in blood donation camps and in case of an emergency. However, it is not recommended as a routine test in blood banks since weakly reactive antigens on cells on forward grouping and low titer anti-A and anti-B on reverse grouping may be missed. Also, drying of the reaction mixture at the edges causes aggregation that may be mistaken for agglutination. Results of slide test should always be confirmed by cell and serum grouping by tube method.
Test tube method is more reliable than slide test, but takes longer time and more equipment. For cell grouping, patient’s saline-washed red cells are mixed with known antiserum in a test tube; the mixture is incubated at room temperature, and centrifuged. For serum grouping, patient’s serum is mixed with reagent red cells of known group (available commercially or prepared in the laboratory), incubated at room temperature, and centrifuged (See Table). Following centrifugation, a red cell button (sediment) will be seen at the bottom of the tube. Cell button is dislodged by gently tapping the base of the tube and examined for agglutination.
 
Positive (+) Test
 
Clumps of red cells suspended in a clear fluid. Agglutination in tube test is graded from 1+ to 4+ and read macroscopically (See Figure). 
 
Grading of ABO tube test
Grading of ABO tube test. Negative: Uniform suspension of red cells; Grade 1 (1+): Many small clumps of red cells (fine granular appearance); Grade 2 (2+): Many large clumps with many free red cells; Grade 3 (3+): Three or four individual clumps with few free red cells; and Grade 4 (4+): One solid clump of red cells with no free red cells
 
Negative (–) Test
 
Uniform suspension of red cells.

Separate tubes of auto-control, positive control, and negative control should always be setup along with the test sample tube. Auto-control tube consists of mixture of patient’s red cells and patient’s own serum. This is required to rule out false-positive result due to auto antibodies in patient’s serum causing auto agglutination of patient’s own red cells. Auto-control test is particularly essential when ABO grouping is being done only by forward method and blood group is typed as AB. If there are auto antibodies in recipient’s serum, ABO grouping, Rh typing, antibody screening, and cross matching all will show positive result.
 
In two positive control tubes, anti-A serum is mixed with group. A red cells and anti-B is mixed with group B red cells respectively. In two negative control tubes, anti-A serum is mixed with group B red cells and anti-B serum is mixed with group. A red cells respectively. These controls are necessary to confirm that reagents are working properly.
 
Interpretation of forward (cell) and reverse (serum) grouping
Interpretation of forward cell and reverse serum grouping
 
Why test tube method of blood grouping is more reliable than slide method?
 
Test tube method of blood grouping is more reliable than slide method. This is because centrifugation enhances the reaction by bringing antigen and antibodies closer together and allows detection of weaker antigen antibody reactions; in addition drying is avoided and smaller amounts of reagent are required.
 
If forward grouping, reverse grouping, and autocontrol tests are all positive, then these results are probably indicative of a cold-reactive autoantibody. Before performing forward typing, red cells should be washed with normal saline to elute the antibody. Before performing reverse grouping, autoantibody should be adsorbed by washed cells till autocontrol is negative.
A WBC differential count gives us information regarding the proportion and numbers of individual leukocytes in the patient’s sample, including significant morphological changes. This can provide useful diagnostic information in cases of inflammation, infection, and antigenic responses.

METHOD
 
Equipment
 
Stained PBS, microscope with 100×objective lens and cell counter.
 
Procedure
 
It is important that examinationand counts be performed withinthe monolayer area of your slide
 
  1. Scan the slide in a methodical grid pattern, in order not to cover the same area twice. Counts can be completed quickly under 400×magnification, but if you are also evaluating morphology, 1000×magnification should be used.
  2. Count a minimum of 100 WBCs.
 
(If the total WBC Count is increased, 200 cells should be counted to maintain accuracy.)
 
Calculations
 
Relative count:
 
No. of Cell Type Seen = ___%
100
 
Absolute count:
 
Relative (%) x WBC Count (10³/ L) = ___ x 10³/μL
100
 
Note: Check your math:
 
• Relative counts of each cell type should add up to equal 100
• Absolute counts of each cell type should add up to equal your WBC count.

Erythrocyte (Gr. erythros, red; kytos, cell) or red blood corpuscles are circular, anucleated, highly flexible, biconcave disc-shaped cells with high edges. The sixe of each cell averages 7.2 micrometer in diameter and 2.1 micrometer in thickness. It is 1.0 micrometer thick in the center. A complex membrane surrounds it, which is a bimolecular layer of protein. There is an inner most structure, called stroma, which is composed of lipids and proteins in the form of a fibrous protein. The cell contents are 90% hemoglobin. There are two methods for estimation of erythrocyte count:

  • Manual or microscopic method
  • Automated method

MANUAL METHOD

Equipment

Hemocytometer with cover glass, compound microscope.

Reagent

Hayem’s diluting solution is prepared as follows:

  • HgCl2 0.05 gm
  • NaSO4 2.5 gm
  • NaCl 0.5 gm
  • Distilled water 100 ml

Specimen

EDTA anticoagulated venous blood or blood obtained by skin puncture is used.

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Method

  1. Wipe finger with cotton soaked with alcohol, with a sterile lancet do small prick on the finger tip. Use pipette. Aspirate blood to 0.5.
  2. Aspirate diluting Hayem’s solution to the 101 mark. It will give 1:200 dilution of the blood.
  3. Hold the pipette horizontally and role it with both hands between finger and thumb.
  4. Place the counting chamber, absolutely free from dust and grease, on the table and lay the cover glass in place over the ruled area.
  5. Discard the first two or three drops from the pipette. Charge the counting chamber by holding the pipette in an inclined position. Allow 3 minutes for the cells to settle.
  6. Locate the central square, which is divided into 25 medium sized squares. Each of the medium sized squares is further divided into 16 smallest squares.
  7. Count the erythrocytes in medium sized squares (80 smallest squares) using high power objective.
  8. In order to avoid confusion in counting, count all cells wihich touch the upper and left outer double line of the group of 16 squares as if they were inside the square. Neglect all those cells, which touch the lower and right inner line.

Calculation

You may calculate total number of erythrocytes per cu mm of the blood as shown in the following.

Supose number of erythrocytes counted in 5 intermediate squares

= E
 
Area of each of the five squares in which cells are counted
 
= 1/25 sq mm
 
Therefore, total area counted
 
= 1/25 sq mm x 5
= 1/5 sq mm
 
Depth of chamber = 1/10 mm
 
Therefore, the volume in which cells are counted
 
= Area x Depth
= 1/5 sqmm x 1/10 mm
= 1/50 cu mm
 
Now, in 1/50 cu mm of diluted blood, the number of erythrocyte counted = E
 
Number of erythrocyte in one cu mm in diluted blood = E x 50
 
Since the dilution of the blood is 1 in 200, the number of erythrocytes in one cu mm of undiluted blood
 
= E x 50 x 200
 

GENERAL NOTES


(1) Increased in numbers of RBC called polycythemia it is due to
 
Congenital heart disease
• Cor pulmonale
Dehydration
• Pulmonary fibrosis
• Polycythemia vera
 
(2) Decreased in numbers of RBC is due to
 
• Anemia
Bone marrow failure
• Erythropoietin deficiency (2ndry to kidney disease)
Hemolysis (RBC destruction) from transfusion reaction
Hemorrhage
• Leukemia
• Multiple myloma
• Nutritional deficiencies of (Iron, Copper, Folate, Vit B12, B6)
 

REFERENCE RANGES

  • Newborns 4.8-7.2 millions
  • Children 3.8-5.5 millions
  • Adult (Male) 4.6-6.0 millions
  • Adult (Female) 4.2-5.0 millions
  • Pregnancy slightly lower than normal

Principle

Anticoagulated whole blood is centrifuged in a capillary tube of uniform bore to pack the red cells. Centrifugation is done in a special microhematocrit centrifuge till packing of red cells is as complete as possible. The reading (length of packed red cells and total length of the column) is taken using a microhematocrit reader, a ruler, or arithmetic graph paper.

Equipment

  1. Microhematocrit centrifuge: It should provide relative centrifugal force of 12000 g for 5 minutes.
  2. Capillary hematocrit tubes: These are disposable glass tubes 75 mm in length and 1 mm in internal diameter. They are of two types: plain (containing no anticoagulant) and heparinised (coated with a dried film of 2 units of heparin). For plain tubes, anticoagulated venous blood is needed. Heparinised tubes are used for blood obtained from skin puncture.
  3. Tube sealant like plastic sealant or modeling clay; if not available, a spirit lamp for heat sealing.
  4. Microhematocrit reader; if not available, a ruler or arithmetic graph paper.

Specimen

Venous blood collected in EDTA (dipotassium salt) for plain tubes or blood from skin puncture collected directly in heparinised tubes. Venous blood should be collected with minimal stasis to avoid hemoconcentration and false rise in PCV.

Method

  1. Fill the capillary tube by applying its tip to the blood (either from skin puncture or anticoagulated venous blood, depending on the type of tube used). About 2/3rds to 3/4ths length of the capillary tube should be filled with blood.
  2. Seal the other end of the capillary tube (which was not in contact with blood) with a plastic sealant. If it is not available, heatseal the tube using a spirit lamp.
  3. The filled tubes are placed in the radial grooves of the centrifuge with the sealed ends toward the outer rim gasket. Counterbalance by placing the tubes in the grooves opposite to each other.
  4. Centrifuge at relative centrifu-gal force 12000 g for 5 minutes to completely pack the red cells.
  5. Immediately remove the tubes from the centrifuge and stand them upright. The tube will show three layers from top to bottom: column of plasma, thin layer of buffy coat, and column of red cells.
  6. With the microhematocrit reader, hematocrit is directly read from the scale. If hematocrit reader is not available, the tube is held against a ruler and the hematocrit is obtained by the following formula:
Length of red cell column in mm
-------------------------------------------------------
Length of total column in mm

To obtain PCV, the above result is multiplied by 100.

GENERAL NOTES

  1. Prolonged application of tourniquet during venepuncture causes hemoconcentration and rise in hematocrit.
  2. Excess squeezing of the finger during skin puncture dilutes the sample with tissue fluid and lowers the hematocrit.
  3. Correct proportion of blood with anticoagulant should be used. Excess EDTA causes shrinkage of red cells and falsely lowers the hematocrit.
  4. Inadequate mixing of blood with anticoagulant, and inadequate mixing of blood before testing can cause false results.
  5. Low hematocrit can result if there are clots in the sample.
  6. Centrifugation at lower speed and for less time falsely increases PCV.
  7. A small amount of plasma is trapped in the lower part of the red cell column which is usually insignificant. Increased amount of plasma is trapped in microcytosis, macrocytosis, spherocytosis, and sickle cell anemia, which cause an artifactual rise in hematocrit. Larger volume of plasma is trapped in Wintrobe tube than in capillary tube.
  8. As PCV requires whole blood sample, it is affected by plasma volume (e.g. PCV is higher in dehydration, and lower in fluid overload).
  9. Expression of PCV: Occasionally, PCV is expressed as a percentage. In SI units, PCV is expressed as a volume fraction. Conversion factor from conventional to SI units is 0.1 and from SI to conventional units is 100.
  10. Rules of 3 and 9: These rules of thumb are commonly used to check the accuracy of results and are applicable only if red cells are of normal size and shape.
    Hemoglobin (gm/dl) × 3 = PCV
    Red cell count (million/cmm) × 9 = PCV
  11. Automated hematocrit: In automated hematology analyzers, hematocrit is obtained by multiplying red cell count (in millions/cmm) by mean cell volume (in femtoliters).

REFERENCE RANGES

  • Adult males: 40-50%
  • Adult females (nonpregnant): 38 45%
  • Adult females (pregnant): 36-42%
  • Children 6 to 12 years: 37-46%
  • Children 6 months to 6 years: 36 42%
  • Infants 2 to 6 months: 32-42%
  • Newborns: 44-60%

CRITICAL VALUES

  • Packed cell volume: < 20% or > 60%

Principle

Anticoagulated whole blood is centrifuged in a Wintrobe tube to completely pack the red cells. The volume of packed red cells is read directly from the tube. An advantage with this method is that before performing PCV, test for erythrocyte sedimentation rate can be set up.

Equipment

  1. Wintrobe tube: This tube is about 110 mm in length and has 100 markings, each at the interval of 1 mm. Internal diameter is 3 mm. It can hold about 3 ml of blood.
  2. Pasteur pipette with a rubber bulb and a sufficient length of capillary to reach the bottom of the Wintrobe tube.
  3. Centrifuge with a speed of 2300 g.

Specimen

Venous blood collected in EDTA (1.5 mg EDTA for 1 ml of blood) or in double oxalate. Test should be performed within 6 hours of collection.

Method

  1. Mix the anticoagulated blood sample thoroughly.
  2. Draw the blood sample in a Pasteur pipette and introduce the pipette up to the bottom of the Wintrobe tube. Fill the tube from the bottom exactly up to the 100 mark. During filling, tip of the pipette is raised, but should remain under the rising meniscus to avoid foaming.
  3. Centrifuge the sample at 2300 g for 30 min (To counterbalance a second Wintrobe tube filled with blood from another patient or water should be placed in the centrifuge).
  4. Take the reading of the length of the column of red cells.

Hematocrit can be expressed either as a percentage or as a fraction of the total volume of blood sample.

Significance

In anemia, PCV is below the lower level of normal range. PCV is raised in dehydration, shock, burns, and polycythemia.

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After centrifugation of anticoagulated whole blood, three zones can be distinguished in the Wintrobe tube from above downwards-plasma, buffy coat layer (a small greyish layer of white cells and platelets, about 1 mm thick), and packed red cells. Normal plasma is straw-colored. It is colorless in iron deficiency anemia, pink in the presence of hemolysis or hemoglobinemia, and yellow if serum bilirubin is raised (jaundice). In hypertriglyceridemia, plasma appears milky. Increased thickness of buffy coat layer occur if white cells or platelets are increased in number (e.g. in leukocytosis, thrombocytosis, or leukemia). Smears can be made from the buffy coat layer for demonstration of lupus erythematosus (LE) cells, malaria parasites, or immature cells.

Packed cell volume (PCV) is the volume occupied by the red cells when a sample of anticoagulated blood is centrifuged. It indicates relative proportion of red cells to plasma. PCV is also called as hematocrit or erythrocyte volume fraction. It is expressed either as a percentage of original volume of blood or as a decimal fraction.

USES OF PCV

  • Detection of presence or absence of anemia or polycythemia
  • Estimation of red cell indices (mean cell volume and mean corpuscular hemoglobin concentration)
  • Checking accuracy of hemoglobin value (Hemoglobin in grams/dl × 3 = PCV).

There are two methods for estimation of PCV: macro method (Wintrobe method) and micro method (microhematocrit method). Micro method is preferred because it is rapid, convenient, requires only a small amount of blood, capillary blood from skin puncture can be used, and a large number of samples can be tested at one time.

This method is also more accurate as plasma trapping in red cell column is less.

By this method, the approximate value of hemoglobin is estimated. This method is simple and rapid. This method is most common in the blood bank for the selection of blood donors.

In this method, a drop of the blood sample is allowed to fall in the solution of copper sulfate having specific gravity 1.053 from the altitude of 1 cm. The hemoglobin concentration of 12.5 g/dl is equivalent to the specific gravity of 1.053. The drop of blood gets covered with copper proteinate and remains separate and distinct for 15-20 seconds. If the drop of blood sample sinks within 15-20 seconds, the specific gravity of copper sulfate solution is lower than the specific gravity of blood sample and the approximate value of hemoglobin is more than 12.5 grams/dl and hemoglobin level is acceptable for the donation of blood. If the drop of blood sample floats, hemoglobin value is less than 12.5 grams/dl and unacceptable for blood donation. However, the concentration of plasma proteins and total leukocyte count also influence the specific gravity of whole blood which may lead false-positive result. In the existence of hypergammaglobulinemia (e.g. multiple myeloma) or leukocytosis (e.g. myeloid or lymphoid reaction, chronic myeloid or lymphocytic leukemia), hemoglobin level will be misleadingly high.

For the estimation of hemoglobin by oxyhemoglobin method, blood sample is mixed with a weak ammonia solution and then absorbance of this solution is deliberated in a photometer using a yellow-green filter or measured in a spectrophotometer at 540 nanometer. Absorbance of the test sample is corresponded with that of the standard solution.For the estimation of hemoglobin by oxyhemoglobin method, blood sample is mixed with a weak ammonia solution and then absorbance of this solution is deliberated in a photometer using a yellow-green filter or measured in a spectrophotometer at 540 nanometer. Absorbance of the test sample is corresponded with that of the standard solution.

This method is much similar to cyanmethemoglobin (hemoglobin-cyanide) method.

This method is very simple and rapid but this method is not much reliable as compared to cyanmethemoglobin method because there is no stable standard solution is available, derivatives of hemoglobin except oxyhemoglobin are not measured, and color of oxyhemoglobin solution swiftly dims.

The major cells of the immune system are lymphocytes. Lymphocytes that are critical for immune reactions are of two types namely B-cells and T-cells. Both cells develop from stem cells located in the liver of the foetus and in bone marrow cells of adults.
 
The lymphocytes which are differentiated in the bone marrow are B-cells. The lymphocytes that migrate to thymus and differentiate under its influence are called T-cells. The young lymphocytes migrate to lymphoid tissues such as spleen, lymph nodes and tonsils where they undergo final maturation. Matured lymphocytes circulate in the body fluids. T-cells are responsible for cellular immunity and B-cells produce antibodies about 20 trillion per day.
 
Both components require antigens to trigger them into action but they respond differently.
 
Antigens
 
An antigen is a substance when introduce into an individual, stimulates the production of an antibody with which it reacts. Antigens are large molecules of proteins or polysaccharides. Some of the antigens are the parts of microorganisms others include pollen, egg white, certain fruits, vegetables, chicken, feathers etc.

Antibodies
 
Antibodies are protein molecules called immunoglobulin (Ig). They are produced by lymphocytes. The antibodies inactivate antigens. An antibody  consists of four amino acid chains bounded together by disulphide bonds. Of the four chains two are long, heavy chains and two are short, light chains. All of them are arranged in the shape of the letter ‘Y’. The tail portion of antibody having two heavy chains is called constant fragment (Fc). On the tip of each short arm, an antigen- binding fragment (Fab) is present which specifically hold antigen.
 
Antibody immunity10
 
Based upon the five types of heavy chains, the immunoglobulin's are classified into five major types. Light chains are similar in all types of Immunoglobulin's.
 
TYPES OF IMMUNOGLOBULIN'S
 
lgG is the most important long acting antibody representing about 80% of the antibodies. The second important antibody is IgM. IgA is called secretory antibody, found in tears, saliva and colostrum, (the first milk secreted by mother). IgD serves as a receptor site at the surface of B cells to secrete other antibodies. IgE plays an important role in allergic reactions by sensitizing cells to certain antigens.
 
iimmunoglobulin types12
Lizard, bird and rabbit, all these three animals are included in group Amniota (Amniote). They excrete the unwanted nitrogenous waste products from their body, and the process is called excretion. If the process of excretion does not take place properly in the body, they become poisonous. In Vertebrates, main excretory organs are called as kidneys. Skin, gills, lungs, liver and intestine are also acts as accessory excretory organs.
 
Kidneys are located on the dorsal side of the coelom and they are made up with numerous uriniferous tubules.
 
Typical, uriniferous tubule is consist of three parts.
 
  1. Ciliated peritoneal funnel
  2. Malpighian body
  3. Ciliated convoluted tube
 
EXCRETORY SYSTEM - GARDEN LIZARD EXCRETORY SYSTEM - PIGEON EXCRETORY SYSTEM - RABBIT
1. Paired kidneys are dark red and irregular in shape. These are flattened organs. 1. Kidneys are dark red and somewhat rectangular and flattened organs. 1. Kidneys are dark red and bean-shaped organs.
2. Kidneys are located in the posterior region of the abdominal cavity and attached to the dorsal wall by a fold of peritoneum. 2. Kidneys are situated in the anterior part of the abdomen. 2. Kidneys are located in the posterior part of the abdominal cavity.
3. Right and left kidneys are opposite to each other. 3. Same as in calotes. 3. The two kidneys are distinct. The right kidney lies much ahead than the left kidney.
4. They are attached to the dorsal muscles. 4. They are fitted in the hollows of the pelvic girdle. 4. Same as in calotes.
5. They are very near to the median line kidneys are Metanephros type. 5. They are a little away from the median line. Kidneys are Meta nephros type. 5. They are well away from the median line. Kidneys are meta ne phros type.
6. Each kidney has two lobes Anterior lobe is broad and posterior lobe is broad Hilus is absent. 6. Each kidney has three lobes They are anterior, median and posterior lobes. Hilus is absent. 6. Each kidney is a single-lobed structure. Inner side of the kidney has a concave depression is known as the 'hilus'.
7. The two kidneys are united posteriorly forming a V-shaped structure. 7. The two kidneys are separate and do not fuse with each other. 7. The two kidneys are distinct.
8. The two ureters are narrow, thin-walled ducts extending behind from the kidneys to the cloaca, where these open into the urodaeum. 8. Same as in Calotes. 8. The ureters open into the urinary bladder. Ureters arise from the hilus of each kidney.
9. There is no pelvis. 9. There is no pelvis. 9. Each ureter is expanded in its kidney into a funnel like pelvis.
10. In males the ureters join at its posterior end with its corresponding vas deferens and both open by a common urino-genital aperture. 10. The ureters do not join with the vas deferens and both open separately into the cloaca. 10. Ureters open separately into the urinary bladder.
11. A thin walled urinary bladder opens on the ventral side of cloaca. 11. Urinary bladder is absent. 11. Urinary bladder is a large, median, pear, shaped, thin walled transparent sac.
12. Urinary bladder communicates with urodaeum thrumph its ventral wall. 12. __ 12. Urinary bladder opens into the urethra or unnogenital canal.
13. Calotes is uricotelic animal Urine consists n.ainly of uric acid. 13. Urine consists mainly of uric acid cotelic animal. 13. Urine consists mainly of urea - ureotelic animal.
14. Urine is excreted in a semi solid state. 14. Urine is excreted in a semisolid state (Bird droppinos). 14. Urine is passed out in a fluid state.
Kidneys are the major excretory organs in all vertebrates. Some other organs such as lungs, gills, liver, intestine and skin also remove certain waste materials besides their normal functions. These are also known as the accessory excretory organs. Both shark and frog are anamniotic animals.
 
The kidneys lie dorsal to the coelom and are composed of large number of renal or uriniferous tubules. A uriniferous tubule typically con­sists of three regions - a ciliated peritoneal funnel, a malpighian body and a ciliated convoluted tube. The malpighian body is a two layered cup, the 'Bowman's capsule' containing a mass of capillaries the 'glomerulus'. The convoluted tube opens into a Longitudinal duct which extends backwards and opens into the cloaca. The excretory organs remove the nitrogenous waste products formed during the metabolic activities from time to time. If these products are not removed from the body, they are changed to toxic substances.
 
EXCRETORY SYSTEM OF FISH EXCRETORY SYSTEM OF FROG
1. Paired kidneys are very long and ribbon like. 1. Paired kidneys are short and roughly oval in shape.
2. Each kidney is differentiated into a small non-renal part (genital part) and a long posterior renal part. The two parts exhibit morphological difference. 2. Each kidney possesses genital as well as renal region. But these are not morphologically differentiat­ed.
3. The kidneys are uriniferous 'Opisthonephros' but functional Mesonephros. 3. The kidneys are mesonephros.
4. Some uriniferous tubules retain peritoneal funnel. 4. The peritoneal funnels are absent.
5. The uriniferous tubules have a specialised urea - absorbing seg­ment. 5. The urea-absorbing segment is absent.
6. Uriniferous tubules lead into special tubes - the urinary ducts (ureters). These are distinct from wolffian ducts. 6. Uriniferous tubules lead into the wolffian ducts.
7. Ureters run back ward over the ventral surface of the kidneys. 7. Wolffian ducts leave outer border of kidneys and run backward.
8. Ureters are independent ducts to carry urine from the kidneys to the Urinogenital sinus'. 8. The ureters serve for the passage of genital elements as well as urine. So they are known as urino-genital ducts.Urino genital sinus is absent.
9. The urinary bladder is absent. 9. A large bilobed urinary bladder is present. It opens into the cloaca opposite the openings of the ureters.
10. The urine is hypotonic to blood. 10. The urine is hypertonic to blood.
11. Scoliodon is an ureotelic animal. The endproduct of nitrogen metabolism is urea. A large Quantity of urea is retained in the body as an adaptation to marine life.Excess of urea is excreted chiefly through its gills. 11. Frog is also ureotelic animal. It excretes urine from the cloaca in the form of urea.
Calotes is a cold blooded (poikilothermic) and terrestrial garden lizard. Pigeon is a ward blooded bird adapted for aerial mode of life. Rabbit is warm blooded and a herbivorous mammal which is also known as Oryctolagus. The circulation of blood in vertebrates is of closed type(circulation occurs is blood vessels. The blood vessels which collect blood from different parts of the body are called as veins. The walls of veins are thick and possess valves.Thier lumen is wide. They collect deoxygenated blood from different parts of the body and carry to the heart. The veins are formed by means of capillaries in the respective tissues or organs. The deoxygenated blood is received by the sinus venosus or the right auricle. The portal veins are having capillaries at their both ends. The pulmonary veins possess oxygenated blood.
 
VENOUS SYSTEM OF CALOTES (GARDEN LIZARD) VENOUS SYSTEM OF COLUMBA (PIGEON) VENOUS SYSTEM OF ORYCTOLAGUS (RABBIT)
1. The venous system consists of common pulmonary vein, two precaval and one post caval veins. These collect blood from the various parts of the body. 1. The venous system con­sists of three large veins-teeo precavak and one post caval along with four large pulmonary veins. 1. The venous system con­sists of four distinct divisions. i) System of venae carae ii) Hepatic portal system iii) Pulmonary system iv) Coronary system
2. The two precaval veins collect blood from the anterior part of the body. Each precaval is formed by the union of the internal and external jugular veins from head and the sub clavian vein from the arm. Transverse jugular vein is absent. Azygous vein is also absent. 2. The two precaval veins collect blood from the anterior part of the body. Each precaval vein is formed by the union of Jugular (head), brachial (arm) and pectoral (Pectoral muscfes) veins. Transverse jugular vessel is present in between the jugular veins. Azygous vein is absent. 2. The two precaval veins collect blood from the anterior part of the body. Each precaval vein is formed by the union of the external jugular vein (head) and subclavian vein (fore limb). The right precaval vein receives the azygous (unpaired) and intercostal veins (intercostal muscles and dorsal wall of theory). Left azygous vein is absent.
3. The post canal vein joins the posterior angle of the sinus venous. It forms by the union right and left efferent renal veins and brings blood from the posterior side. 3. The post caval vein is formed by the union of two large itac veins a tittle behind the liver. 3. The post caval vein is a large median vein. It stands at the cauda region (icaudal vein) and runs forward and receives blood in its course. The veins which join the posl caval vein are pairec ilio himbars, iliacs gonadial renal, anc hepatic.
4. The renal portal system collects blood from the posterior side of the body. Caudal vein bifurcates into two pelvic veins which . unite in front and form into the median anterior abdominal vein enters into the liver. Each pelvic vein joined by femoral, sciatic veins of that side. From the pelvic arise the renal portal veins which branch into capillaries in the substance of the kidneys coccygeo-mesenteric vein is absent. 4. Renal portal system is not well developed in pigeon caudal vein bifurcates into right and left renal portal veins (Hypo gastric veins) each of which enters the kidney. The hypogastric vein receives the Internal iliac vein abng with femoral & sciatic veins. At the bifurcation of the caudal vein into the two renal portal veins arise a median 'coccygeome-senteric vein'. It is characteristic of birds. The coccygeo- mesenteric vein joins the hepatic portal vein. 4. Renal portal system is completely absent in Rabbit.
5. The Hepatic portal vein collects blood from the alimentary canal and enters the liver and breaks upto capillaries. 5. The Hepatic portal vein collects bbod from the alimentary canal and emptied into the liver. From the Ever the blood is carried by the post caval vein through hepatic veins. 5. Same as in pigeon.
6. Epi gastric vein is absent. 6. Epi gastric vein returns the blood from the mesenteries and joins the hepatic veins. This vein corresponds to the abdominal vein of the frog. 6. Epi gastric vein is absent.
7. The right and left pulmonary veins bring pure blood from the right and left lungs and united into a common branch. Common pulmonary vein opens into the left auricle. 7. Four large pulmonary veins return blood from the posterior part of the left auricle. 7. A pair of pulmonary veins bring oxygenated blood from the lungs They unite by a common arch and open into the dorsal wall of the left auricle.
8. The right auricle receives deoxygenated blood through sinus venosus and left auricle possess oxygenated blood. In the partially divided ventricle the blood mixes to some extent. 8. The right side of the heart (right auricle & ventricle) receives de-oxygenated blood and left side folded with (left auricle & ventricle) oxygenated blood. 8. Same as in pigeon. Coronary veins collect deoxygenated blood from the wall of the heart. The coronary sinus opens into the right auricle through an aperture guarded by the Valve of The besius'. The opening is called as the 'formina of the The besius'.
Scoliodon commonly called as shark fish is a poikilothermic (cold blooded) animal. It is cartilaginous fish. Rana (frog) is also poikilothermic and amphibious animal. The circulation of blood in vertebrates is of closed type. The blood vessels which collect blood from various parts of the body are known as veins. The walls of the veins are thin and possess valves. Their lumen is wide. They collect deoxygenated blood from different parts of the body and carry to the heart. The veins are formed by means of capillaries in the respective tissues or organs. The deoxygenated blood first enter into the sinus venosus which is the part of the heart. The portal veins are having capillaries at their both ends. The pulmonary veins possess oxygenated blood.
 
 
FISH (SHARK) - VENOUS SYSTEM FROG (RANA) - VENOUS SYSTEM
1. The venous system comprises a system of large thin walled sinuses which collect blood from the different body organs 1. The venous system comprises of thin walled tubular veins.
2. It consists of the following systems i) Anterior cardinal system ii) Posterior cardinal system iii) Hepatic porta! system iv) Ventral veins vi) Cutanecious system 2. It is divided into i) Anterior system of veins ii) Posterior system of veins iii) Portal systems.
3. The anterior cardinal system and the interior jugular sinuses collect blood from the head region through a number of sinuses. 3. The blood from the head region is collected by a pair of precoval veins. Each precaval vein is formed by External jugular, innominate and subclavian veins.
4. The blood from gills is collected by five pairs of dorsal nutrient sinuses and five pairs of ventral nutrient sinuses. 4.The blood from the lungs is collected by a pair of pulmonary veins.
5. The nutrient sinuses carry deoxygenated blood. 5. The pulmonary veins carry oxygenated blood.
6. The nutrient sinuses empty into anterior cardinal and interior jugular sinuses which inturn open into the ductus cuvieri. Thus the blood finally carried to the sinus venosus. 6. The pulmonary veins open into the left auricle.
7. From the posterior part of the body the blood is collected by i) a pair of posterior cardinal sinuses ii) a pair of lateral abdominal veins iii) a pair of brachial veins. 7. The blood from the posterior part of the body is collected by i) renal portal system and ii) Post caval vein.
8. The renal portal system includes the caudal vein and the renal postal veins & Iliac veins. The blood from the pelvic fins is not carried to the kidneys. 8. The renal portal system consists of veins hind limbs i.e. femoral, sciatic and renal portal veins. The caudal vein is absent.
9. It is absent. 9. A part of the blood from the hind-body is transported to the liyer by an anterior abdominal vein.
10. The blood from the kidneys is collected by renal veins which open into posterior cardinals, opening into the cuvierian sinus. 10. The blood from kidneys is collected by four pairs of renal veins which open into the post caval vein.
11. The brachial veins join the lateral abdominals to form sub clavian veins which open into the ductus cuvieri. 11. The brachial veins open into the precaval veins particularly into the subclavian veins.
12. Three pairs cutaneous veins collect blood from the muscles of skin and open into the cardinal sinuses, lateral abdominals and brachial veins. 12. The cutaneous veins are only one pair which join with muscular & brachial and finally open into the subclavian veins.
13. The venus blood does not enter the sinus venosus directly. But it is collected first by the cuvierian sinus present transversely. 13. The blood collected by the two precavals and one post caval veins directly enters into the sinus venosus.
14. The blood from the parts of the alimentary canal is collected by the Hepatic portal vein and empties into the liver and from there it is transported by Hepatic sinuses into the sinus venosus. 14. The Hepatic portal vein collects blood from the different parts of the alimentary canal and empties into the liver. From the blood is transported into the sinus venosus through the hepatic veins and post caval vein.
Calotes is  called as garden lizard. It is a poikilothermic terrestrial reptile. Columba is commonly called as pigeon, being a bird it is  adapted for aerial mode of life. Oryctolagus (rabbit) is an herbivorous mammal. The blood circulation in these vertebrates is of closed type. The blood vessels which carry blood from the heart to the various parts of the body are known as arteries The walls of the arteries are thick and do not possess valves. The pure blood flows in the arteries. However the arteries which carry blood from the heart to the respiratory organs possess deoxygenated blood. The blood has high pressure in the arteries. Arteries ends by means of blood capillaries in the tissues. The different arteries associated in the circulation of blood form a system which is called as Arterial System.
 
In the above three vertebrates, the arteries arise differently but carry blood from the heart to the various parts of the body.
 
 
Calotes (Garden Lizard) Columba (Pigeon) Oryctolagus (Rabbit)
1. Arterial system consists of a pair of systemic arches and a pulmonary arch. 1. Arterial system consists of two arches-Right sys­temic arch and pulmonary arch. The right systemic arch is called Right Aortic arch. 1. Aiterial system con­sists of two arches, left systemic arch and pulmonary arch. The left systemic arch is known as Left aortic arch
2. The systemic arches and the pulmonary arch arise from the dorsal and ventral parts of the single ventricle. All the three arches are connected by connective tissue. 2. Right aortic arch arises from the left ventricle and pulmonary arch arises from the right ventricle. 2. Left aortic arch airses from the left ventricle and the pulmonary arch arises from the right ventricle.
3. The carotid branch of each side is connected with systemic arch by a vessel, 'Ductus caroticus'. 3. Ductus caroticus is absent. 3. Same is absent.
4. The subclavian arteries arise from the right systemic arch. 4.The right & left carotid and sub clavian arteries originate from the respective innominate arteries, ises. 4. The right carotid and sub clavian arteries arise from the innominate artery. But the left carotid and sub calvian arteries originate directly from the right aortic arch
5. Inter cestal arteries are present. 5. Same are present. 5. Same are present.
6. It is absent. 6. Pectoral artery supplies blood to the muscles of the wings. 6. It is absent.
7. Coeliac artery and anterior mesenteric artery arise separately from the dorsal from the dorsal aorta. 7. Same in pigeon. 7. Same in rabbit.
8. It is absent. 8. It is absent. 8. Phronic artery supplies blood to the muscles of the Diaphragm.
9. A pair of gonadial arteries are present. 9. From the anterior renal arteries the gonadial arteries are formed. 9. Paired Gonadial arteries arise directly from the dorsal arch.
10. Unpaired posterior mesenteric artery is present. 10. Same is present in pigeon. 10. Same is present in rabbit.
11. Three pairs of renal arteries arise from the dorsal aorta. 11. The anterior renal arteries develop from the dorsal aorta. But middle & posterior renal arteries arise from the sciatic artery of each side. 11. A pair of renal arteries arise from the dorsal aorta.
12. Common Iliac ar­teries are formed from the dorsal aor­ta. 12. The internal iliac arteries are formed from the dorsal aorta. 12. Iliolumbar arteries arise from the dorsal aorta.
13. The caudal artery is the terminal portion of the dorsal aorta to the tail. 13. The caudal artery the terminal portion of the dorsal aorta to the tail. 13. The caudal artery is the continuation of the dorsal aorta to the tail.
14. The pulmonary arteries carry blood from right part of the single ventricle to theright and left lungs. 14. Each pulmonary artery carries deoxygenated blood to the respective lung for purification. 14. The pulmonary artery which arises from the right ventricle divides into two branches and carry deoxygenated blood to the respective lungs.
15. Coronary arteries supply blood to the walls of the heart. 15. Coronary arteries supply blood to the walls of the heart of bird. 15. Coronary arteries supply blood to the walls of the heart.
Scoliodon ( Shark) is a poikilothermic animal. It is a cartilaginous fish. Frog ( Rana) is a cold blooded and amphibious animal. The circulation of blood is carried by closed vessels. The vessels which supply blood to the various organs of the body are known as arteries as the net work of arteries form the Arterial system. The walls of arteries are thick and lumen is narrow. The blood pressure is high in the arteries. Arteries do not possess valves. The arteries end in capillaries. Arteries deeply seated in the body. Mostly arteries contain oxygenated blood. A few arteries also carry deoxygenated blood to the respiratory organs (either gills or lungs) for purification.
 
 
Scoliodon (Fish) Rana (Frog)
1. The arterial system consists of a ventral aorta, afferent and efferent branchials, dorsal aorta, and its branches and hypobranchials. 1. The arterial system consists of a truncus arteriosus, three pairs of aortic arches and the dorsal aorta & its branches.
2. Five pairs afferent branchial arteries are present. 2. Absent.
3. Efferent branchial system is associated with gill-slits along with the respective arteries. 3. Absent.
4. The arteries to the head are given off from the first pair of epibranchials and by the branches of dorsal aorta carotid labyrinth is absent. 4.The head is supplied blood by the branches. Carotid arteries arising from the truncus arteriosus. Carotid labyrinth is present.
5. Parietal arteries are present. 5. Parietal arteries are absent.
6. Hypobranchial plexus is present. 6. It is absent.
7. Dorsal aorta is formed by the union of epibranchial arteries of both the right and left sides. 7. The second branches of turncus, the systemic arches of the left and right sides unite to form the dorsal aorta.
8. Subclavian arteries arise from the dorsal aorta. 8. Sub clavian artery arises from each systemic arch.
9. Absent. 9. Occipito-vertebral artery arises from the systemic arch of each side.
10. Coeliaco-mesenteric artery aris­es from behind the union of the four pairs of epibranchials. 10. Coeliaco-mesenteric artery arises from the junction of the two system¬ic arches.
11. Just below the Coeliaco-mesen­teric artery, lienogastric artery arises. 11. Lie no gastric artery is absent.
12. The parietal artery gives off four pairs renal arteries. 12. Four pairs of renal arteries arise directly from the dorsal aorta.
13. Gonadial (Spermatic or ovari­an) artery arises from the lieno ­gastric artery. 13. Gonadial arteries arise directly from the first pair of renal arteries.
14. Dorsal aorta terminates into caudal artery. 14. C-iudal artery is absent.
15. Pulmo cutaneous arch is absent. 15. The third branch of truncus is the pulmo-cutaneous arch which is divided into pulmonary and cutanecious arteries.
 
The heart of fish possess venous blood and blood passes through the heart only once in a complete circuit. But in frog the heart receives both oxygenated and venous blood and the circulation is bi circuit.
 
The fish is an aquatic animal and possesses five pairs of gills. The blood is supplied by pairs of afferent bronchial arteries and is collected by nine pairs of efferent bronchial arteries. In frog however, the respiratory organs are a pair of lungs (skin & buccal cavity also help in respiration) which are supplied by a pair of pulmonary arteries.
Calotes is a poikilothermic terrestrial lizard. Columba is pigeon adapted for aerial mode of life. Oryctolagus is an herbivorous mammal.
 
Both pigeon and Rabbit are warm blooded animals. Heart, arteries, veins and blood capillaries are included in the circulatory system. The blood circulation is controlled by an important organ Heart. Normally the blood flows in the closed vessels. So blood circulation is of closed type in verte­brates. The heart possesses auricles and ventricles. The pericardium is at­tached to the heart by gubernaculum cordis'.
 
The number of chambers of heart varies from calotes and other two vertebrates (Pigeon & Rabbit). The heart contracts and relaxes rhythmi­cally. This is called heart beat.
 
The detailed comparison of the heart of the above three animals is mentioned below.
 
Calotes (Lizard) Columba (Pigeon) Oryctolagus (Rabbit)
1. Heart is situated mid ventrally in the an­terior part of the body cavity in the pleuro peritoneal cavity be­hind the sternum. 1. Same way the heart is located. 1. Heart is situated in the thoracic cavity, between the lungs of two sides (Mediastinum). It is present slightly towards the left side.
2. Heart is comparatively smaller in size. 2. Heart is comparatively larger in size. 2. Heart is comparatively larger in size.
3. It is enclosed by double walled pericardium. 3. It is also enclosed in the double walled pericardium. 3. Same.
4. Heart includes a dorsal sinusvenusus aright auricle, a left auricle and a single incom-pletely divided ventricle. 4. Heart is four chambered, sinus venosus is absent in the adult. Completed divided two auricles and two ventricles by inter auricular septum and inter ventricular septum respectively. 4. Same as in columba.
5. The three vena cavae or two precavals and a post caval vein open into the sinus venosus. 5. The three vena cavae or two precavals and a post caval empty the blood directly into the right auricle. 5. Same as in pigeon.
6. The left auricle receives two pulmonary veins from the lungs. 6. The left auricle receives four pulmonary veins from the lungs. 6. Left auricle receives two pulmonary veins from the lungs.
7. The right auricle pos­sess sinu auricular aperture guarded by valve. 7. Absent. 7. Absent.
8. The two auricles are completely separated by inter auricular sep­tum. But the inter ven­tricular septum in the ventricle is incomplete. Hence oxygenated and deoxygenated types of blood is mixed to some extent in the ventricle. 8. Complete inter auricular and inter ventricular septa are present. There is no possibility of mixing the oxygenated blood with deoxygenated blood. 8. Same as in pigeon.
9. The heart of lizard is in a transitional stage approcarhing the double circuit stage But it has not reached it completely due to incomplete division of the encircle. 9. The heart is a double circuit heart because of complete division of ventricle into right and left chambers. 9. Same as in pigeon.
10. The auriculo ventricular aperture is guarded by two flap like semilumar valves. 10. The right auriculo ventricular aperture is guarded by two large muscular flap like valve and the left by three valves. 10. The right auriculo-uentricular aperture is guarded by tricuspid valve and the left by bicuspid valve (mytral valve).

11. There are three aortic arches arising from the ventricle.

  1. Pulmonary trunk (ventral most)
  2. Right systemic trunk (arise from left side of ventricle)

11. Only two aortic arches originate from the ventricles.

  1. Pulmonary trunk (from right ventricle)
  2. Right systemic trunk (from left ventricle)

i.e. Right aortic arch is characteristic of birds.

11. Only two aortic arches arise from the ventricles.

  1. Pulmonary arch (right ventricle)
  2. Left systemic aorta (Left aortic arch from the left ventricle). Right aortic arch is absent.
12. Ductus caroticus is present (connection between carotid & systemic arches) 12. Absent 12. Absent
13. Lizard's heart presents a transitional heart, since it approaches the double circuit heart but has not yet completely attained. So the heart is less efficient. 13. Avian heart has at tained maximum com olexity and is a double circuit heart, i.e. venous blood is com pletely separated frorr oxygenated blood. 13. Same as in Pigeon.
14. Absent. 14. Sinu-Auricular Node and Auriculo ventricular node are present. 14. SA - node and A.V. node are present. In addition bundle of His muscles are also develop.
Scoliodon is a poikilothermc and cartilagenous fish. Rana is also poikilothermic and amphibious animal. In the circulatory system the heart is the most important organ. The blood vascular system in the vertebrates is of closed type. The heart lies in the pericardial cavity of the coelom. It is on the ventral side of the alimentary canal and present anteriorly. In scoliodon the heart is two chambered where as in Rana it is three chambered.
 
Heart is a pumping organ of blood. From various parts of the body it collect blood mainly through veins and supplies blood through arteries.
 
Normally the heart is enclosed by a double walled pericardium which possess pericar­dial fluid. The heart contracts and relaxes rhythmically which facilitate the circulation of blood.
 
FISH HEART (SCOLIODON) FROG HEART (RANA)
1. Heart is approximately pear-shaped. 1. Heart is approximately pear-shaped.
2. The pericardial cavity is not wide and the pericardium forms double membrane around the heart. 2. The pericardial cavity is not wide and the pericardium forms double membrane around the heart.
3. The heart is formed of a dorsally placed sinus venosus and ventrally placed two auricle, a ventricle and truncus arteriosus or conus arteriosus. 3. The heart is formed of a dorsally placed sinus venosus and ventrally placed two auricle, a ventricle and truncus arteriosus or conus arteriosus.
4. The atrium or auricle is two-chambered and lies anterior to the ventricle. Auricles are separated by Inter auricular septum. 4. The atrium or auricle is two-chambered and lies anterior to the ventricle. Auricles are separated by Inter auricular septum.
5. The auriculo-ventricular valve is membranous. 5. The auriculo-ventricular valve is membranous.
6. The conus arteiiosus is incompletely divided by the spiral valve laterally into cavurn aorticum leading to carotid and systemic arches and the cavum pulmocutaneum leading to the pulmocutaneous arch. 6. The conus arteiiosus is incompletely divided by the spiral valve laterally into cavurn aorticum leading to carotid and systemic arches and the cavum pulmocutaneum leading to the pulmocutaneous arch.
7. The opening of the truncus with valves are arrenged in two transvarse rows. 7. The opening of the truncus with the ventricle is guarded by three semilunar valves arranged in a single row. They devide rruncus into a proximal pylangium and a distal synangium.
8. The walls of the auricle are thick. 8. The muscular walls of the auricle are thin.
9. The walls of the ventricle are highly muscular. 9. Same type of ventricle is present.
10. The lips of the bilaminate valves are connected to the inner surface of the ventricle is prominent part of the heart. 10. The membranous valves are connected to the inner surface of the ventricle by chordae-tendinae. Both auricles and ventricle are essential parts of the heart.
11. The fish heart is venous or branchial heart because it receives deoxygenated blood only. 11. The frog's heart receives both oxygenated and deoxygenated blood. The deoxygenated blood remain separate in the auricles but get mixed in the ventricle.
12. Blood passes only once through the heart in a complete circuit. 12. Blood passes through the heart twice in a complete circuit.
13. Such type of arrangement is absent. 13. The sinus venosus opens into the right auricle through simi-auricular aperture guarded by simi auricular valve which is also known as pace maker.
14. No separate vessel collects oxygenated blood since the heart is venous heart 14. The oxygenated blood is collected by pulmonary vein from lungs and carries into left auricle.
The Appendicular skeleton is one of the divisions of the endo skeleton. It includes the pectoral and pelvic girdles and limb bones. The skeleton of the limb in all the tetrapods shows a similar fundamental and structural similarity. However the differences such as arms, legs, wings and paddles are seen in the respective animals. A few tetrapods have completely lost one or both pairs of appendages. The limbs are totally absent in caecilians, most snakes and snake-like lizards.The Appendicular skeleton is one of the divisions of the endo skeleton. It includes the pectoral and pelvic girdles and limb bones. The skeleton of the limb in all the tetrapods shows a similar fundamental and structural similarity. However the differences such as arms, legs, wings and paddles are seen in the respective animals. A few tetrapods have completely lost one or both pairs of appendages. The limbs are totally absent in caecilians, most snakes and snake-like lizards.

The typical tetrapod hind limb can be divided into three seg­ments. The thigh, shank and foot (pes) are the three segments. If there are five toes, normally this type of limb is known as pent dactyl limb.
 
The skeletal structures of the hind-limb consists of femur, tibia, fibula, tarsals, metatarsals and phalanges.

The femur is the bone of the high and its head articulates with the acetabulum. Its distal end articulates with fibula. The tibia and fibula are the bones of the shank region. They articulate with femur proximally and distally with the tarsal’s of the ankle bones. The fibula bears the most of the body weight.

The foot can be divided into ankle, instep and toes. The ankle is supported by tarsals, which are arranged in rows. The skeleton of ankle or tarsus is the most stable of the regions of the ankle. The instep or metatarsus is supported by the metatarsals. These are elongated bones. The metatar­sals are followed by linear series of phalanges of the toes. The phalanges number varies from 1 to 5.

The first toe of the hind limb is called 'hallux or great toe' and the fifth toe is the 'minimus'.
 
Calotes (Garden Lizard) Columba (Pigeon) Oryctolagus (Rabbit)
1. The bones of the hind limb are femur, Tibia, fibula, tarsals, metatarsals and phalanges. 1. The bones of the hind limb are femur, tibia, fibula, tibiotarsus, tarsometatarsals and phalanges. 1. The bones of the hind limb are femur, tibia fibula tarsals, meta tarsals and phalanges.
2. The femur is stout bone of the thigh region. It has long, slender and curved shaft in the middle. The shaft enlarges at both the ends. 2. The femur is a stout bone of the thigh region. It has a long, curved shaft in the middle. The shaft has broad ends. 2. Femur consists of long, stout curved shaft. The femur gives support to the thigh region.
3. The proximal end of the shaft bears a rounded smooth head which fits info the acetabulum. There are also distinct prominences lesser trochanter and greater trochanter near the head. 3. The proximal end of femur is produced into a rounded head for the articulation with the acetabulum. Opposite to the head a small protuberance greater trochanter is present. 3. The proximal end of femur bears a rounded knob-like head which fits into the acetabulum. There are three rough projections greater, lesser and third trochanters present near the head. Lesser trochanter lies behind the head, greater trochanter in the middle line and the third trochanter opposite to the head are seen.
4. It is absent. 4. There is an articular surface is present between the head and trochanter for the antitrochanter of ilium. 4. It is absent.
5. Two knob-like condyles are present at the distal end of thefemur. These articulate with the tibia of the shank. Intercondylar groove is present between the two condyles. Patella is absent. 5. The distal end of femu has two prominent condyls with a intercondylar groove. Patella slides in the intercondylar groove on the anterior side. It is a disc-like sesmoid bone. 5. The distal end of femur is pulley-like having two condylesfor tibio-fibule which are separated by a patellar groove. A large sesmoid bone called the patella slides in the patellar groove. It is attached to the tibia by a ligament. Patella is present at the knee-joint.
6. The shank consists of two long bones - the tibia and the fibula. They are separate bones. 6. Tibiotarsus fibula is formed of tibiotarsus and fibula. They are separate bones. 6. Tibiofibula is formed of tibia and fibula. They are separate bones.
7. Tibia is a stout and curved bone present on the inner side. Its proximal end bears two concave facets for the articulation with the femur. It has also a longitudinal ridge the cnemial crest on the side. Tibiotarsus is absent. 7. Tibiotarsus is a large straight and stout bone and also longer than fibula. It is formed by the fusion of tibia and proximal row of tarsals. The proximal end of it bears a pair of articular surfaces for the condyles of the femur and in between them the cnemial crest for the attachment of tendon of extensor muscles. 7. Tibia is stouter towards the anterior end and narrow towards the posterior end. Its proximal end bears two concave facets for the articulation with the femur and distinct cnemial crest on side.
8. Tibia distally bears a concavity for the tarsals. 8. Tibio tarsus distally bears a pulley-like articular surface for the tarsals which is surrounded by a pair of distal lateral tubercles. 8. Tibia distally bears articular surface for the tarsals.
9. Fibula is a slender bone present on the outerside. It bears facets on either side. 9. Fibula is small, slender bone. It is closely applied to the tibiotarsus. 9. Fibula is a slender and weak bone. It lies on the outer side. The bone is narrower towards the distal end and is closely applied to the tibia.
10. Tarsab are five in number which are arranged in two rows. Proximal row has two tarsals the larger compound piece formed by the fusion of a rjbiale, intermedium and centrale and present infront of tibia. A small fibulare present infront of the Sbula. The distal row has three small tarsab called distal tarsab or distalia. 10. The free tarsals are absent. The proximal row of tarsals are fused with tibia and forms tibiotarsus. The distal row of tarsals are fused with the metatarsals and forms tarso metatarsus. It is as long as the femur bone. It is straight and stout. 10. There are six tarsal bones which are arranged in two rows. The proximal row tarsab are two, astragalus and calcaneum. Astragalus is considered to represent two fused tarsals. Calcaneun is produced back wards into a strong calcaneal process which forms the heel. The central row has only one tarsal-centrale or navicular. The distal row contains three tarsab. The first distal tarsal is absent due to the absence of hallux. The second distal tarsal is mesocuneiform which is the smallest distal tarsal. The third distal tarsal is ecto cuneiform which largest one. The fifth distal tarsals are fused to form largest bone in the row - cuboid.
11. There are five meta-tarsals corresponding to the five toes. 11. There are four meta tarsals. The first one is free and in the form of a small projection. The second, third and fourth are fused with the distal row of tarsals to form tarso metatar­sus. Ankle joint is known as mesotarsal. 11. There are four meta tarsals. There are second, third, fourth and fifth, meta tarsals. The first one is absent
12. There are five toes. There are two pha­langes in the hallux, three in the second, four in the third, five in the fourth and three in fifth toes. The pha­langes formula can be expressed as 2, 3, 4, 5, 3 (same as for the hand). The terminal phalanx of each toe supports a strong, curved, horny & pointed claw. 12. There are four toes. The hallux is directed backwards and contain two phalanges. The second toe with three, third one with four and the fourth one with five phalanges are formed. The phalanges formulae can be ex¬pressed 2, 3, 4, 5. The terminal phalanx of each toe is pointed and curved which supports a strong, pointed horny claw. 12. There are four toes. Each toe has three phalanges. The phalanges for­mula can be ex­pressed as 3, 3, 3, 3. The terminal part of each phalanx is pointed and curved to support a horny claw.
The Appendicular skeleton is one of the divisions of the endo skeleton. It includes the pelvic and pectoral  girdles and limb bones. The skeleton of the limb in all the tetra pods shows a fundamental and structural similarity. However, the differences such as arms, legs, wings and paddles are seen in the respective animals. A few tetra pods have completely lost one or both pairs of appendages. The limbs are totally absent in caecilians, most snakes and snake-like lizards. In sirens, the lizard-chirotes, manatees and dugongs only fore-limbs are present. The Appendicular skeleton is one of the divisions of the endo skeleton. It includes the pelvic and pectoral  girdles and limb bones. The skeleton of the limb in all the tetra pods shows a fundamental and structural similarity. However, the differences such as arms, legs, wings and paddles are seen in the respective animals. A few tetra pods have completely lost one or both pairs of appendages. The limbs are totally absent in caecilians, most snakes and snake-like lizards. In sirens, the lizard-chirotes, manatees and dugongs only fore-limbs are present.

A typical tetrapod fore limb can be divided into three segments. The upper arm, fore arm and hand (menus) are the three segments. As there are five fingers normally, this type of limb is known as pentadactyl limb.
The skeletal structures of the fore limb consists of humerus, radius ulna, carpals, Meta carpals and phalanges.

The humerus is the bone of the upper arm and its head articulates with the glenoid cavity .Its distal end articulates with the ulna The Radius and ulna are the bones of the fore arm. They articulate with humerus proximally and distally with the carpals of the mist bones. The radius bears most of the body weight.

The hand can be divided into wrist, palm and digits (fingers). The wrist is supported by carpal bones which are arranged in rows. The palm is supported by the metacarpals. The metacarpals are followed by linear series of phalanges of the fingers The phalanges number vary from 1 to 5.

The first finger of the fore limb is called 'pollex or thumb' and the fifth finger is the 'minimus'.
 
Calotes (Garden Lizard) Columba (Pigeon) Oryctolagus (Rabbit)
1. The bones of the fore limbs are humerus, radius, ulna, carpals, metacarpals and phalanges. 1. The bones of the fore limb are humerus, radius, ulna, carpals carpometa carpus and phalanges. 1. The bones of the fore limb are humerus, radius, ulna, carpals, metacarpals and phalanges.
2. Humerus is in the form of a long bone with proximal and distal ends. 2. Humerus is a long & slightly flattened with a bent shaft associated by proximal and distal ends. 2. Humerus possess a proximal head, shaft and a distal end.
3. The proximal end of humerus is round and distal end is pulley like with two articular surfaces for the radius and ulna. Supra trochlear foramen is absent. 3. The proximal end of humerus is highly expanded and form into the head A prominent deitcid ridge and a pneumatic foramen are present near the head. The distal end articulates with the radius and ulna by the articular surfaces. Supra trochlear foramen is absent. 3 The proximal end of hu-merus is divided into two parts by a bicipital groove. One part has head which fits into the glenoid cavity. This part has lesser tuberosity. The greater tuberosity is present on the other part. Shaft is present along with deltoid ridge. The distal end has median and lat¬eral epicondyles. Pulley-like trochlea is formed at the distal end which articulates with ulna. Suprotrochlear foramen is present.
4. Two elongated and separate radius and ulna bones are present. 4. Same as in calotes. 4. Same as in columba.
5. Radius is a slender bone. It has a styloid process and concavity for the carpalsdistally. 5. Radius is a straight and slender bone. It has a concavity for the articulation with humerus at the proxima end The distal end is convex. 5. Radius is small slen­der and slightly curved bone With a concavity at the proximal end. The distal end is flat.
6 Ulna is rod liket and stoutet than radius, Proximally it has ole cranon process to articulate with humerus. Distally it has a concavity for the articulation with carpals. 6. Ulna is stouter and longer than radius It is slightly curved. A cranon process to blunt olecranon process is present at the proximal end. The distal ends of radius & ulna articulate with carpometacarpus. 6. Ulna is a long and curved bone. Proximally it bears olecranon process and sigmoid notch for the articulation with the trochlear end of humerus. Epiphyses are present at the distal ends of radius & ulna for the articulation with carpals.
7. Wrist or carpus has ten (10) small bony carpals arranged in three rows. The proximal row has three carpals - radiale, intermedium and ulnare. A centrale lies in the second row. A pisi­form is attached to the distal end of the ulna on its post axi­al side as an addi­tional bone. The third row has five distal carpals. Ex­cept the fourth, the remaining distal carpals are very small. 7. The wrist contains only two proximal carpals. One smaller-radiate and a larger ulnare articulate with radius & ulna respec­tively. The three dis­tal carpals are fused with the meta carpa­ls to form the carpometa carpus. It is a characteristic feature of aves. 7. Wrist consists of eight small carpal bones arranged in two rows. The proximal row contains three carpals-radiale or scaphoid, intermedium or semilunar and ulnare or unciform. The median row has a single centrale. The distal row comprises four true carpals-trapezium, trapezoid, smallest magnum and largest unciform.
8. Carpometa carpus is absent Five slen­der meta carpals support the palm. These are of unequal size & with expanded ends. The middle or third meta carpal is the longest, the second and fourth are only a little shorter than the third. The first and fifth meta carpals are much shorter. 8. Meta carpals are three in number which are fused with the distal carpals and form an elongated compound bone carpometa carpus. 8. There are five long, slender and of unequal size metacarpals support the palm. The first is the shortest and the third is the longest. Each meta carpal has small epiphysis at their end with a middle slender shaft. Carpometa carpus is absent.
9. There are five fingers. 9. There are three fingers. 3. There are five fin­gers.
10. The phalanges are the small bones sup­port the fingers. The number of phalanges differ in the respec­tive fingers. The first finger has two, sec­ond has three, third has four, fourth has five and fifth has three phalanges Thus the phalanges formula can be ex­pressed as 2,3, 4, 5, 3. 10. The phalanges are the small bones sup­port the fingers. The first finger has one, second has two and third has one phalan­ges. Thus the phalan­ges formula can be expressed as 1, 2, 1.There are no claws on the fingers. 10. The phalanges are small bones and their total number is 14. The first finger has two phalanges & the remaining four fingers have three phalanges each. Thus the phalanges formula can be expressed as 2,3,3,3.3.
11. Sesamoid bones are absent. The distai phalanx of each finger supports a strong curved, pointed claw is formed from the epidermis. 11. Sesamoid bones are absent. 11. Sroas nodule-like bones are present on the underside of the fingers. These are seen at the joints between the meta carpals and the first phalanges and also between the second and third phalanges. These provide additional strength to the fingers during burrowing.
12. It is a penta dactyl limb. 12. The fore limb supports the wing. 12. it is a penta dactyl limb.
The hard parts of the animal body are collectively known as skeletal system or simply skeleton. The vertebrates possess the hard parts inside the body. It is known as endo skeleton. The endo skeletal structures are formed with cartilages and bones which are the living tissues. The endo skeleton has been divided into:The hard parts of the animal body are collectively known as skeletal system or simply skeleton. The vertebrates possess the hard parts inside the body. It is known as endo skeleton. The endo skeletal structures are formed with cartilages and bones which are the living tissues. The endo skeleton has been divided into:
 
  1. Axial skeleton - includes the skull and vertebral column.
  2. Appenducular skeleton - includes the girdles and limb bones.
 
The skull develops in the head of animal body. The skull includes two major parts - 'Cranium' enclosing the brain and the organs of special sense and Visceral arches' which form the jaws and frame work of pharyngeal wall.

The cranium is developed from the mesodermal cells soon after the appearance of the brain. It is also known as brain box. Cranium includes three pairs of capsules for smell, sight and hearing. These are known as olfactory, optic and auditory capsules respectively. The cartilaginous cranium is called chondro cranium and bony cranium is called dermato cranium.

The visceral arches develop around anterior (Pharyngeal) part of the embryonic gut from the cells of neural crests. Mostly seven visceral arches are present. The first one is the largest and highly modified - 'Mandibular arch. It has dorsal & ventral halves. Each side of the dorsal half is termed the palato -pterygoid Quadrate Cartilage. It bears teeth and forms the upper jaw. The ventral half of the mandibular arch is called Meckel's cartilage. It also bears the teeth and form the lower jaw. The wide gap between the two jaws is the mouth. The two jaws articulate their hind ends by hinge joints which enable the mouth to open & close. The second arch is hyoid arch and the remaining five arches are termed bronchial arches. The visceral arches are collectively known as the splanchno cranium. The upper jaw and lower jaw are known as Maxilla and Mandible respectively: See images. 
 
SKULL OF SCOLIODON (Shark) SKULL OF RANA (Frog) SKULL OF CALOTES (Garden Lizard) SKULL OF COLUMBA (Pigeon) SKULL OF ORYCTOLAGUA (Rabbit)
1. Skull is formed with cartilage tissues. 1. Skull is formed most­ly with bony tissues (but tadpole skull is cartilaginous) 1. Skull is formed most­ly with bony tissues. 1. Skull is formed mostly with bony tissue. 1. Skull is formed with mostly bony tissue.
2. It consists of crani­um, sense capsules and visceral arches. 2. It consists of cran­ium, sense capsules, jaws and hyoid ap­paratus. 2. It consists of crani­um, sense capsules, jaws and hyoid apparatus. 2. Same as in calotes. 2. Same as in calotes.
3. It is the axial portion of the skull. It is more or less a violin box open in front and be­hind with an arched roof and flattened floor. It is divided into occipital, auditory, orbital and ethmoidal regions. 3. It forms the middle hollow part of the skull. It is divided into auditory, olfactory and occipital regions. 3. It forms the median hollow part of the skull. It is divided into occipital, audi­tory, orbital, olfacto­ry and optic regions. 3. It forms the posterior median hollow part of the skull. It is divided into occipital, audito­ry, optic orbital and ol­factory regions. 3. It forms the middle hollow part of the skull. It is divided into occipital auditory, optic orbital & olfac­tory regions.
4. Foramen magnum is posteriorly present. 4. Same. 4. Same. 4. Same. 4. Same.
5. Beneath the foramen magnum a deep concavity is present. On either side of this concavity is a pro­minence - occipital condyle articulates with the first verte­bra, occipital crest is formed. Dicondylic skull. 5. Beneath the foramen magnum there are two occipital con­dyles. On either side of the foramen mag­num dorsolaterally exoccipital bones are present. Dicondylic skull 5. Beneath the fora­men magnum a sin­gle occipital condyle is present.suupraoccipitai, exo occipitals,& basi occipital bones are also present in the occipital region. Monocondylic skull. 5. Beneath the foramen magnum single occip­ital condyle is present. Supra occipital, Exocci pitals & basioccipital bones are also present. Monocondylic skull. 5. Beneath the fora­men magnum two occipital condyles with paroccipital process are present. Supraoccipital, exo-ccipitai, & basio-ccipital bones are also present. Dico­ndylic skull.
6. Auditory region has a mid dorsal depres­sion - parietal fossa. It contain two pairs of apertures. Anteri­orly smaller open­ings of endolymp­hatic ducts and pos­teriorly larger open­ings of perilymphatic spaces are present. 6.— 6.— 6.— 6.—
7. Auditory capsules lie on the poster lat­eral sides of the cranium. Which enclose & protect the ears. Post orbital groove is present on the ven­tral side 7. Auditory capsules enclose the internal ear. Its roof is formed by pro-otic bone, fenestra ovalis, sta­pedial plate and columella auris are present. 7. Each auditory capsule is formed by small, single vertical prootic bone which is lying outside the supra occipital. Epiotic & opisthotic are not differentiat­ed. 7. Each auditory capsule is formed largely by the prooticbone. Fenestra ovalis, fenestra rotun da, columella auris, stapes are also present. 7. Each auditory cap­sule in the adult animal consists only periotic. Flask - like Tympanic bulla bone is significant.
8.— 8. Dorsally the cranium is formed, by frontoparietals, ven­trally by parasphenoid and laterally by sphen ethmoid bones. 8. The dorsal part of the cranium is formed by parietals, frontals interparietal foramen, and ven­trally by basisphenoid, parasphenoid bones. 8. The dorsal part of the cranium is formed by Parietals, frontals a rostum, alisphenoids; ventrally basisphenoid, basitemporal bones. 8. The cranium is formed dorsally by 'Parietals, frontals, inter parietal, and ventrally by basisphenoids, presphenoid bones along with alisphenoids and orbit sphenoids. The cra­nial cavity is closed infront by a narrow vertical bone cibriform plate.
9.  Each orbit lies on the sides of the middle part of the cranium. It is bordered by dor­sal super orbital ridge,anterior preorbital process, posterior post orbital process and ventraily by infra orbital ridge. The orbital region has a large oral cavity anterior fontanelle. 9. On either side of the cranium is large gap - orbit which lodges the eye. 9. In the middle of the cranium laterally two orbits are present. Each orbit is bounded by prefrontal supra orbital, lacri­mal, post frontal and jugal bones. The jugal bone forms the ventral border of the orbit. Supratemporal arch is present. 9. The two orbits are very large cavities present infront of the cranium. Each orbit is bounded dorsally by frontal, antero - dorsally by lac­rimal and posteriorly by the zygomatic process. Orbit is incomplete on the ventral side. The two orbits are separated by inter orbital septum. 9. These are two orbits are large sockets present on the sides of frontal segment of cranium. The orbit is bounded dorsally by frontal, anteriorly by maxilla and lacrimal, posteriorly by squa­mosal and alisp-henoid and external­ly by the zygomatic arch.
10. The olfactory cap­sules lie at the anteri­or side of the cranium. Each capsule possesses a short sic at ethmopalatine ridge. 10. The olfactory cap­sules are separated, from each other by mesethmoid. Each capsule is formed by a large triangular nasal on the dorsal side and a smaller triradiate vomer on the ventral side vomers possess vomerine teeth. 10. Each olfactory capsule is formed by three bones Nasal, septo maxillary and vomer. 10. Each olfactory capsule is formed by two bones - Nasal and vomer. Nasals fuse with frontals and form into super and inferior processes. 10. Each olfactory cap­sule is bounded by dorsally by long na­sal bone and laterally by jaw bones. The two capsules are sep­arated by mesethmoid bone. The lower end of mesethmoid fits into a vomer bone. Vomer is formed by the fusion of a pair of bones.
11. Ethmoidal region tapers anteriorly. It consists of a basal slender barventro-median rostral carti­lage and a pair of similar barsdorso - lateral rostral cartilages aris­en from the roof of ihe olfactory capsules. 11. Absent. 11. Absent. 11. Absent. 11. Absent.
12. Scoliodon has seven visceral arches which are cartilagienous. The first arch forms the jaws and it is catted Mandibular arch the second one is the hyoid arch the remain­ing five arches are called branchial arch­es. 12. Branchial arches are absent.There are upper and lower jaws to support the borders of the mouth. The upper jaw is formed by union of two similar halves. Each half is formed by the Pre-maxilla, maxilla and quadratojugal. The inner set of the jaw has palatine, ptery goid and squamosal bones. The lower consists of two halves and unite an­teriorly by mento-meckelian cartilage. Each half consists of dentary and angio -splenial bones. Just infroni of the articu­lar fact a small coro-nary process is present. Upper jaw alone has teeth. 12. Branchial arches are absent. 12. Branchial arches are absent. 12.   Branchial arches absent.These are upper and lower jaws. Each half of the upper jaw is formed by premax-illa, maxilla jugular, palatine, pterygoid and squamosal.
13. The mandibular arch consists of two halves. Each half of this arch possess an upper paleto-pterygo quadrate cartilage and a lower meckel s cartilage.The pale topterygo Quadrate gives off anteriorly palatine. The two sides of it from the upper jaw with teeth. The two meckel's cartilages united antero medially by lig­ament form the lower jaw with teeth.   13. These are upper and lower jaws. Each half of the upper jaw consists of an outer set of bones - pre maxilla, maxilla, jugal and quadrate and the inner set in­cludes pterygoid, palatine, transp-alatine, epiptery-goid and squamo­sal. Each half of the lower jaw consists of six bones -dentary, angular, supra angular, ar­ticular, splenial and coronoid. Both the jaws possess teeth. 13. These are upper and lower jaws. Each half of the upper jaw is formed by premaxilla, maxilla, quadra tojugal, and jugal bones. The inner ar­cade of the upper jaw forms the roof of bucco pharyngal cav­ity which consists of palatine, pterygoid, and quadrate. Each half of the lower jaw is formed by articu­lar, angular supra an­gular, dentary and splenial. Both the jaws are lacking the teeth. 13. The lower jaw also con­sists of two halves. Each half is formed by a single, large dentary bone. The posterior of the dentary possess con­dylar, coronoid and angular process. Both the jaws pos­sess the codent type of teeth which are having different (Heterodont teeth in mammals) shap­es. Diastema is present in both the jaws because of the absence of canines.
14. Hyostylic jaw suspension. 14. Auto stylic jaw suspension. 14. Auto stylic jaw suspension. 14. Auto stylic jaw suspension. 14. Craniostylic jaw suspension.
The pelvic girdle is directly attached to the vertebral column in the sacral region. The pelvic girdle consists of two similar halves which are known as ossa innominata. Each os innominatum is. formed by three bones. The dorsal bone is known as ilium, antero-Ventral bone is named as pubis and the ventral bone is called ischium. The pelvic girdle has a depression (concavity) at the junction of the three bones. It is known as acetabulum, into which the head of femur of the hind limb articulates.
 
bird pelvic girdle18
 
The same bones are present in all the pelvic girdles of the different vertebrates but have undergone modification.
 
 
rabbit pelvic girdle16
 
Calotes (Garden Lizard) Columba (Pigeon) Oryctolagus (Rabbit)
1. Pelvic girdle is stout and solid. Ifts well suited for walking habits. 1. Pelvic girdle is large and pneumatic. It is well suited for bipedal locomotion. 1. Pelvic girdle is stout and associates with the vertebral column. It is adopted for swift running.
2. Each os innominatum is formed by the ilium, ischium and pubis. 2. Same as in calotes. 2. Each os innominatum is formed by ilium, ischium, pubis and cotyloid cartilage bones.
3. The bones are structurally united. 3. The bones are compactly fused. 3. Same as in columba.
4. Ilium is strong, rod shaped and is directed upwards. 4. Ilium is long, thin and plate-like bone. It is differentiated into preacetabular and postacetabular regions. 4. Ilium is large and broad. The antero-dorsal edge is raised to form iliac crest.
5. Ilium articulates with two sacral vertebrae. 5. Ilium articulates with synsacrum. 5. Anteriorly ilium has articular surface for the sacral vertebrae.
6. Ischium is flat, slightly curved and axe-shaped. It is directed downwards and backwards. 6. Ischium a flat bone fused with the post acetabulariiium. They are separated by ilio-ischial foramen. 6. Ischium is broad and slightly curved bone lying behind ilium. It is posterodorsal in position.
7. Ilio-ischial foramen is present. 7. llio-ischial foramen is large. 7. Ilio-ischial foramen is absent.
8. Ischial tuberosity is absent. 8. It is absent is pigeon. 8. Ischium bears an ischial tuberosity.
9. Ischial symphysis is present. 9. It is absent. 9. Ischial symphysis is present.
10. Hypoischium is present between the ends of the two ischia. 10. Absent. 10. Absent.
11. On the ventral side posteriorly the pubis is formed like a flat elongated and slight ly curved bone, pubis. 11. Pubis is long, slender, curved bone. It lies ventral and parallel with ischial, Pubis. 11. Pubis is flat curved bone directed ventrally pubis symphysis is present. Epipubis is absent.
The hip or pelvic girdle' is present in the posterior side of the body to which the pelvic fins or hind limbs are attached. The pelvic girdle is connected directly to the vertebral column in the sacral region. The pelvic girdle has two equal halves which are known as 'ossa innominata'. Each as innominatum is formed by three bones. They are the dorsal bone ilium, the ventral bone-ischium and the antero-ventral bone pubis. The pelvic girdle has a depression at the junction of the three bones. It is termed as acetabulum into which the head femur of the hind limb articulates and forms a ball and socket joint.
 
frog pelvic girdle15
 
In the different vertebrates, the same bones are present in the pelvic girdle with some modifications.
 
 
Shark (Scoliodon) Frog (Rana)
1. The pelvic girdle is formed with cartilage tissues. 1. The pelvic girdle is formed chiefly with bone tissues.
2. It is embeded in the body wall muscles infront of the cloacal aperture. 2. It is present at the hind end of the trunk.
3. It is a simple transverse bar known as ischio - pubis bar. 3. It consists of two similar halves which are separated infront and fused behind to form a median vertical disc.
4. Each half of the girdle is formed by the fusion of ilium ischium and pubis. 4. Each half of the girdle consists of three bones - ilium, isclium and pubis.
5. Acetabulum is absent. 5. Each side of the vertical disc bears a cup-like depression Acetabulum. The head of femur of the thigh bone articulates with the acetabulum. So all the three bones take part in the formation of the acetabulum.
6. The ilium possess an iliac process and a foramen. 6. The ilium extends forwards in the form of an arm to articulate with the transverse process of the sacral vertebra. A vertical ridge is formed along with this arm is called iliac crest.
7. Ischium and pubis fused together and form Ischio-pubis bar. 7. The ischium forms the posterior part of the disc and acetabulum. Ischium, fuses with the other side ischium and forms ischium symphysis.
8. Pubis fuses with ischium. It is not a separate bone. 8. The pubis forms the ventral part of the disc and acetabulum. It fuses with the pubis of the other half and forms pubic symphysis. It is a separate bone.
9. Pubis is formed with cartilage tissue. 9. Pubis is formed with calcified cartilage tissue.
10. The pelvic girdle is straight in the middle but bent at the ends. These are produced dorso-lat-erally into short iliac processes. 10. The pelvic girdle V-shaped associated with a vertical disc formed with the ischium & pubis bones.
11. The pelvic fins are attached directly. 11. The hind limb bones are articulating with the pelvic girdle.
12. The pelvic girdle provides attachment to the claspers through the muscles of male. 12. Such arrangement is absent. Penis is absent.
Page 4 of 17

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