FISH PHYSIOLOGY

Note that:

1. Fish blood clots much rapidly than blood of other animals .so use an anticoagulant treated syringes and needles (EDTA,
heparin , Na citrate or oxalate)

2. Anasthetize the fish before collection through

1. using piece of cotton wetted with methyl alcohol 40% by putting it under operculum cover , within 3 minutes the fish loss
all reflex
2. Immersing the fish in a solution of urethane 0,5 % these solution .this solution is carcinogenic so use plastic gloves for
protection
3. Iiodocaine spray

Methods of blood collection → I. Caudal vein II. Heart III. descending aorta

1. Caudal vein

 From very small fish :cutting the caudal peduncle of fish

 From large fish :using syringe, passing the needle through midline directly behind anal fin of the fish till hit the vertebral
column then suck blood gently

2.Heart behind the opercula cover

 From the ventral at the med line 1cm depth in the back of tail direction with 45 angle in the big fishes
 The puncture should be deeper, he needle is directly moved dorsally until the heart puncture and blood flow
3. Descending aorta

 The method is used in bigger fishes.

 The needle is longer it pass through oral cavity in the direction of the gills in acute angle puncturing should be superficial
and avoid finger pressure to stop bleeding

Erythrocyte count in fish

 As mammalian blood using heamocytometer

 Diluent used: Natt and Herrick's solution as poultry
 Erythrocyte count in fish :2-5million cell /mm3
 Erythrocyte count vary according species, age , season and environmental fish
 Shape :oval , biconcave, nucleated
 Leukocyte count :150.000 cell /mm3
 Major abundant leukocyte in fish :lymphocyte
 Thrombocyte :round oval or spindle , nucleus oval
 ESR:3mm/hrs
 Practical avian hematology

The basic hematology principle of mammalian hematology apply to avian hematology with some modification

Collection of Blood sample

 The total blood volume that can be collected from birds must be not exceed than 10 % of its weight (about 6- 12 ml L100
gm of body weight).
 There are different methods for blood collection by using micro collection devices as needle or syringe with good
attention must be taken to avoid vein collapse or hematoma.

1-Jugular vein puncture:

 In birds right jugular vein is larger than the left one so it must be attempt to puncture firstly by wetting the skin of the
neck with alcohol the blood collected using syringe for vein puncture

2. Wing or Brachial vein:

 This method used in small sized birds and blood collected using special needle to minimize the formation of hematoma.

3. Caudal tibia vein:

 It located on the medial aspect of the leg above the tarsal joint in most birds.

4. Clipping of toenail:

 It applied by cutting the toe and permit blood to flow freely then apply hemostatic agent as ferrous sulphate after blood
collection
  All cellular element of the birds blood (RBCS, WBCS and Thrombocytes) are nucleated
 The erythrocytes of birds are
 Oval shaped and nucleated with a diameter larger than mammals (12X7 um).
 The thrombocytes of bird show considerable variation in size and shape may be vary from oval to round. The typical
plateletes are oval with round nucleus in the center of clear cytoplasm with the presence of one or more red stained
granules at RBCS one pole of the cells.
 The number of platelet in avian blood ranged between 20.000 to 30.000
Experiment (2)

 Avian erythrocytes count:2.5-3.5 million cell /mm3 Anemia polytheciemia

 Theory of the experiment:
 Erythrocytes are counted by using haemocytometer and Natt and Herrick's solution as diluent to RBCS pipette.

 Natt and Herrick's solution

Nacl 3.88 g
NA2SO 4 2.5 g
Na2PO4 2.91 g
Formalin (40%) 7.5 ml
Methyl violet 0.1 g
DW 1000 ml

Hemoglobin

 Hb is large molecule consisting of four individual polypeptide chains, each with a heme (iron containing ) protein that can
binds oxygen in the (Ferrous form) ( Fe++ )
 Hemoglobin of adult male chicken is 12gm/dl however in female is 9.11 gm /dl. V
 In pigeon male hemoglobin level is 15.9 gm however in female one is 14.7 gm
 In duck female is15.6 gm.
 The most reliable method for its determination in birds is cyanomethemoglobin method
Experiment (3) Hemoglobin determination

 The best method for hemoglobin determinations in bines is the method of Denington and Luceas as in the following steps:
1. Fill a test tube with 0.4% solution of ammonium hydroxide.
2. Take up to 20 ul of blood sample by hemoglobin pipette the discharge it into the ammonia solution.
3. Seal the tube with cork and invert the tube several time and leave for one hour.
4. Add 0.36 ml of concentrated Hel to the tube then invert the several times.
5. After formation of precipitate of hemin and hemoglobin bound to this precipitate.
6. The density of color was determined by a colorimeter at wave length 410 nm.
7. The obtained transmission value (R) was multiplayed with 2.3736 to give the number of grams of hemoglobin per 100ml
of blood.

Experiment (4) Osmotic properties of erythrocytes

 The osmotic pressure of avian RBCS are higher than of mammals (equal to 1.02
% NaCl).
 The minimal resistance in birds was 0.4 % NaCl while the maximal resistance
was 0.3 % NaCl.

Avian hematology
 Total leukocytes count in avian:20,000-30,000 cell /mm3
 Leucopenia: decrease of leukocyte
 Leukocytosis; increase of leukocyte
Total leukocytes count in birds can be estimated by

 Direct method using Natt and Herrick's solution

 N.B: mixture used for RBCS counts therefor the total RBCS and WBCS
obtained by counts are the charged same haemocytometer.

 Indirect method can be used after count of RBCS then count the WBCS in five oil immersion field on the blood smear and
apply the in the following formula:

 No. of WBCS / mm3 = No. of cell counted per 5 field X 3500.000/1000

 Where 1000 is the average number of RBCS in 5 field.

 3500.000 is the normal count of RBCS in birds.

Differential leukocytes count:

 Theory of the experiment:

1) Leukocytes of birds have a characteristic feature that differed from mammals.
2) The heterophils are difficult to differentiate from acidophils by the ordinary stain so it
needs special stain as Wright's Leishman and Papnheim stains.
Heterophil:

 This cell type could be known as polymorphncclear or psodoesinophilic cell where the granules of this cell in bird are acidic
in reaction.
 The heterophils of chicken are usually round with rod or spindle shaped esinophilic granules in their cytoplasm.
 The nucleus is polymorphic with varying degree of lobulation.
 It represents about 27 % of the total count of WBC.

Eosinophil:

 The size of eosinophils is the same of heterophils. Their granules are spherical, large and with dull red colour.
 The nucleus is bilobed and with richer blue than that of heterophils.
 It represents about 2% of the count of WBC Basophil; It has the same size as heterophils.
 Their nucleus is oval shape with weakly basophilic characters.
 The cytoplasm is devoid of color and contains deeply basophilic granules.

Lymphocyte:

 It is the predominate cells of avian blood leukocyte as represent about 60% of the WBC count.
 The cytoplasm is weakly basophilic and considered as a narrow rim bordering on one side of the large round nucleus.

Monocyte:

 It is a larger leukocyte with relatively more cytoplasm than lymphocytes.

 The cytoplasm has blue gray color.
 The nucleus is similar to that of lymphocytes with irregular shape in outline with delicate pattern of chromatin
characters. OIt represents 10% of total WBC count.
Experiment (5) Blood group in birds

 Theory of the experiment:

1) In chicken blood groups are different than that reported in mammals. So blood grouping is determined by producing
antigens.
2) There are at least 11 separately inherited blood groups system which are determined by a separated genetic locus.
3) The locus is defined as a site on a chromosome where two or more alternative genes have been shown to occur.
4) From these groups are O, H. I, J, K, L. N or P and within the group presence of sub- group.
5) The blood groups in birds have relation to productivity so the breeders use blood group to predict an excellent
productive trails.