Practical Manual of Comp.Ani.Physiology

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Information about Practical Manual of Comp.Ani.Physiology

Published on July 2, 2017

Author: ravindrakshirsagar

Source: slideshare.net

1. Comparative Animal Physiology/M.Sc. Zoo-I/Modern College G.K page 1 PRACTICAL MANUAL OF COMPARATIVE ANIMAL PHYSIOLOGY As Per the Syllabus Prescribed By SP Pune University ZY 205 P: M.Sc. Zoology (2 credits) Dr. Ravindra V.Kshirsagar M.Sc.Ph.D. F.G.S.B.A.S Post graduate Department of Zoology P.E.S Modern College of Arts, Science and Commerce, Ganeshkhind, Pune. Email: ravindrakshirsagar3@gmail.com For review and recommendation for Private Circulation only

2. Comparative Animal Physiology/M.Sc. Zoo-I/Modern College G.K page 2 Date: Name of the student: Practical No: 1 Title of the Experiment: Study of nitrogenous waste products of animals from different habitats. Signature of Practical in charge: Dr.Ravindra Kshirsagar Practical No: 1

3. Comparative Animal Physiology/M.Sc. Zoo-I/Modern College G.K page 3 Title of the Experiment: Study of nitrogenous waste products of animals from different habitats. Theory: Different animals expel different nitrogenous compounds. On the basis of the type of nitrogenous end product. There are 3 modes of excretion. They are: (a) Ammonotelism :Aquatic habitat (b) Ureotelism :Terrestrial habitat (c) Uricotelism. Terrestrial and Aerial habitat Ammonotelism It is the type of excretion in which ammonia is the main nitrogenous waste material. Such animals are called ammonotetic. Occurrence It is found in aquatic animal groups like sponges, coelentrates, crustaceans, echinoderms, bony fish, tadpole larvae and salamander. Ammonia is produced as a result of catabolism of proteins, especially in the liver cells by oxidative deamination of excess of amino acids in the presence of oxidase enzyme. Ammonia is highly toxic and must be metabolised or expelled from the body as soon as possible. Ammonia is highly soluble in water and a very large volume of water is needed by the animal to dissolve ammonia. 1 gm of ammonia needs about 300 - 500 ml of water. But this is not a problem for animals living in an aqueous habitat which are generally found to be ammonotelic. Ureotelism It is a type of excretion where urea is the main nitrogenous waste material. Animals showing ureotelism are called ureotelic animals. Generally found in land animals which can afford to excrete sufficient volume of water or to concentrate urea in considerable quantity in the urine. It is commonly found in man, whales, seals, desert mammals like kangaroo rats, camels, toads, frogs, cartilagenous fishes, aquatic and semi aquatic reptiles like alligator, terrapins and turtles.

4. Comparative Animal Physiology/M.Sc. Zoo-I/Modern College G.K page 4 In the liver of the animals, ammonia is detoxified to form urea by the orrithine cycle. Urea is far less toxic than ammonia and so can remain inside the body for a longer period without causing any ill effects inside the body. 1 gm of urea needs about 50 ml of water to the expelled out. Uricotelism Elimination of uric acid as the main nitrogenous waste material is called uricotelism. Animals showing uricotelism are called uricotelic animals. Occurrence It is a common method seen in birds, land reptiles, insects, land snails and some land crustaceans. Uric acid is formed from ammonia mostly in the liver and to some extent in the kidneys. The process is highly energy dependant, but is much less toxic than both ammonia and urea and it is almost insoluble in water and can be eliminated from the body in nearly a solid state, saving a lot of water.

5. Comparative Animal Physiology/M.Sc. Zoo-I/Modern College G.K page 5 Test for Urea: 1The testing of urea is performed by taking 10 ml of milk in a test tube and then adds 4 drops of bromothymol blue solution and after 10 minutes if the color of solution remains blue after shaking then solution indicates the presence of urea. 2 The thymolblue solution is a 0.5 % solution of the indicator in ethanol. It has been chosen among many different indicators for it is a 3-color indicator. According to the literature data [3] in a strongly acidic medium the color changes from red (pH < 1.2) to yellow (8 > pH > 2.8), and in a weakly alkaline medium it changes from yellow to blue (pH > 9.6). One uses the above chemicals, a test-tube rack with test-tubes and a dropper. Test for Ammonia: Ammonia: Salicylate test: The ammonia-salicylate method involves a three-step reaction sequence. The first reaction step involves the conversion of ammonia to monochloroamine by the addition of chlorine. The monochloroamine then reacts with salicylate to form 5-aminosalicylate. Oxidation of 5- aminosalicylate is carried out in the presence of a catalyst, nitroferricyanide, which results in the formation of indosalicylate, a blue-colored compound. The blue color is masked by the yellow color (from excess nitroprusside) yielding a green-colored solution that absorbs light at 650 nm. The intensity of the color is directly proportional to the ammonia concentration in the sample. (1) Ammonia compounds are initially combined with hypochlorite to form monochloramine; (2) Monochloramine reacts with salicylate to form 5- aminosalicylate.

6. Comparative Animal Physiology/M.Sc. Zoo-I/Modern College G.K page 6 Test For Uric acid:

7. Comparative Animal Physiology/M.Sc. Zoo-I/Modern College G.K page 7 Date: Name of the student: Practical No: 2 Title of the Practical: RBCs in different vertebrates and in different physiological conditions. Signature of Practical in charge: Dr.Ravindra Kshirsagar Practical No:2

8. Comparative Animal Physiology/M.Sc. Zoo-I/Modern College G.K page 8 Title of the Practical: RBCs in different vertebrates and in different physiological conditions. Introduction to tonicity: Tonicity is a measure of effective osmolality in cell biology. Osmolality is the properties of a particular solution, independent of any membrane. Tonicity is a property of a solution in reference to a particular membrane, and is equal to the sum of the concentrations of the solutes which have the capacity to exert an osmotic force across that membrane. Tonicity, also, depends on solute permeability (permeant solutes do not affect tonicity; impermeant solutes do affect tonicity). Tonicity is generally classified in three ranges; hyper tonicity, hypo tonicity and isotonicity. Hypertonic, isotonic and hypotonic solutions are defined in reference to a cell membrane by comparing the tonicity of the solution with the tonicity within the cell. Figure; 1 Diagram of Hypotonic, Hypertonic and Isotonic solutions effect on red blood cells. Hypertonic:

9. Comparative Animal Physiology/M.Sc. Zoo-I/Modern College G.K page 9 The word "HYPER" means more, so hypertonicity refers to a solution that has a higher or more of a concentration to it's external environment. The cell has a higher number of particles (solutes) dissolved in it than the solution outside of the cell membrane. When a cell’s cytoplasm is bathed in a hypertonic solution the water will be drawn into the solution and out of the cell by osmosis. This causes water to move out of the cell, it shrivels up and shrinks. HOW IT AFFECTS ANIMAL CELLS; Animal Cells In animal cells, being in a hypertonic environment results in crenation, where the shape of the cell becomes distorted and wrinkled as water leaves the cell. Some organisms have evolved methods of venting Hypertonicity; for example, saltwater is hypertonic to the fish that live in it. Since they cannot isolate themselves from osmotic water loss, because they need a large surface area in their gills for gas exchange, they respond by drinking large amounts of water, and excreting the salt. This process is called osmoregulation. An example of an animal cell showing the affects of hypertonicity is when your fingers wrinkle. Figure2;Hypertonicity Hypotonic

10. Comparative Animal Physiology/M.Sc. Zoo-I/Modern College G.K page 10 The word "HYPO" means less, in this case there are less solute molecules outside the cell. Cells hypotonic to their surrounding solutions cause water to move into the cell and cause it to expand. The cell has a smaller number of solutes than the solution outside of the cell membrane. A hypotonic solution is a solution having a lesser solute concentration than the cytosol. It contains a lesser concentration of impermeable solutes on the external side of the membrane. When a cell’s cytoplasm is bathed in a hypotonic solution the water will be drawn out of the solution and into the cell by osmosis. If water molecules continue to diffuse into the cell, it will cause the cell to swell, up to the point that cytolysis (rupture) may occur. The opposite of a hypertonic environment is a hypotonic one, where the net movement of water is into the cell. If the cell contains more impermeable solute than its surroundings, water will enter it. HOW IT AFFECTS ANIMAL CELLS; In an animal cell, hypotonic solutions cause them to swell, they become bloated and triple their original size. By the time they have reached triple their size (how long this takes depends on each individual cell) they burst like a balloon and die.

11. Comparative Animal Physiology/M.Sc. Zoo-I/Modern College G.K page 11 Isotonic Isotonic: A cell in an isotonic environment is in a state of equilibrium with its surroundings. When the amount of impermeable solute is the same on the inside and outside of the cell, osmotic pressure becomes equal; the force of water trying to exit and enter the cell balances out."ISO" means the same meaning that the osmotic pressure and concentration of solutes is the same in both the internal and external environments of the cell. Cells isotonic to their surrounding solutions have an equal concentration of solutes in and out of the cell membrane. This creates a dynamic equilibrium that maintains the status of the cell. No change will occur in the cell.

12. Comparative Animal Physiology/M.Sc. Zoo-I/Modern College G.K page 12 Date: Name of the student: Practical No: 11 Title: Determination of the heart beat in the crab-effect of temperature & ions. Signature of Practical in charge: Dr.Ravindra Kshirsagar

13. Comparative Animal Physiology/M.Sc. Zoo-I/Modern College G.K page 13 Practical No: 11 Title: Determination of the heart beat in the crab-effect of temperature & ions. Anatomy of Crab: Procedure: 1 After obtaining a live crab, freeze at -20ºC for one hour to kill your specimen. For observing anatomy and keep live for Heart observation. Do not leave your specimen in the freezer longer than two hours as internal organs may begin to disintegrate. Rapid freezing with liquid nitrogen is also an option, but take note that it is necessary to allow minimal thawing prior to dissection as the carapace may freeze to the internal organs as a result of ice formation. 2 Determine if your crab is male or female. This is easily done by observing the telson folded under the ventral surface of the specimen. A female has a broad U-shaped telson, while a male has a narrow V-shaped telson. 3 Begin the dissection by carefully removing the carapace. This can be done by using a scalpel to cut around the lateral edges, working from one side, posteriorly, and up the other side. The

14. Comparative Animal Physiology/M.Sc. Zoo-I/Modern College G.K page 14 carapace can be opened as if on a hinge attached at the eye-stalks. Be careful when lifting the carapace, since the epidermis may adhere to the underside of the carapace. 4 Remove the epidermis. The easiest way to do this is under a dissecting microscope using a pair of dissecting tweezers. Be careful when removing the epidermis since rapid removal tends to disturb the underlying hepatopancreas Heart and stomach. Structure of Heart of Crab: The crustacean adult heart is neurogenic (same in Horseshoe crabs), and beats according to inputs from cardiac ganglia as a pacemaker and there is no inherent cardiac heartbeat The heart is dorsal and surrounded by a pericardial sinus.It is single chambered (typically NOT tubular) with one or two pairs of ostia Anterior and posterior leads into several different arteries. Anterior ones include an anterior aorta and others that lead directly to the liver and other organs posterior arteries supply the legs. Heart Regulation of crab: Crab cardiac system. Single-chamber heart (red) is located within the pericardium (not shown), which is flanked by paired neurohemal structures, the pericardial organs (POs; blue). (POs and the nerves linking the heart with the CNS are shown on one side only.) Cardiac ganglion (CG), containing 4 interneurons (purple) and 5 motor neurons (magenta), lies beneath the dorsal surface of the heart and generates the motor patterns that drive the contractions of the heart

15. Comparative Animal Physiology/M.Sc. Zoo-I/Modern College G.K page 15 muscle. Heartbeat is regulated by neurons within the CNS (black) in two ways. Neurons project through segmental nerves such as SN1 (segmental nerve of the first thoracic neuromere, green) to the POs, where they liberate into the pericardial sinus neurohormones that then reach the heart via the blood. In addition, neurons project further through the dorsal nerve (green) to directly innervate the heart and, within it, OBSERVATION: Crab Barytelphusa Cunicularis Collected from local market Yerawada Pune, approximately 4.7 cm. total body length were collected and maintained the laboratory. Conditions for at least one week under natural photo period. The animals were blotted dry and their body weight was recorded. Two different groups of crabs ranging in their body weight from 10 to 12 gm. were selected. While studying the effect of temperature on heartbeat the crabs having uniform body weight and carapace width were selected and kept at temperature at 20O C, 25O C, 30O C, etc. Sr.no Weight of Crab Normal Heart beat/Min. 1 2 Effect of tempreture: Sr.no Weight of Crab Tempreture Normal Heart beat/Min. 1 20O C 2 25O C 3 30O C Conclusion

16. Comparative Animal Physiology/M.Sc. Zoo-I/Modern College G.K page 16 In crustaceans i.e. Crab heart is a single chambered, contraction and relaxation of it are controlled by nerve fiber, hence it is a neu rogenic heart.as the temperature increases the heartbeat also increase.

17. Comparative Animal Physiology/M.Sc. Zoo-I/Modern College G.K page 17 Date: Name of the student: Practical No: EFFECT OF EYE STALK ABLATION ON GLUCOSE CONTENT IN THE HAEMOLYMPH OF THE CRAB Signature of Practical in charge: Dr.Ravindra Kshirsagar

18. Comparative Animal Physiology/M.Sc. Zoo-I/Modern College G.K page 18 EFFECT OF EYE STALK ABLATION ON GLUCOSE CONTENT IN THE HAEMOLYMPH OF THE CRAB Principle: Eye stalk of crab contains an endocrine tissue structure known as X organ. This endocrine structure is neurohaemal made up of group of neurosecretory cell. The axon of these neurosecrtory cell group together and terminates outside vessel. The site of their fusion is known as nurohaemal organ which are endocrine in function. The hormone secreted from eyestalk organ of crab regulates the haemolymph glucose level. The hormone is protienecious in nature. The extract of eye stalk when injected in to crab in aqueous media has shown rapid increase in the concentration of reducing sugar in haemolymph. However injection of sinus gland extracts induces hyper glycaemia in all species. The body fluid sugar content before and after eye ablation can be estimated by acid phenol and anthrone method. In presence of strong acid glucose gets converted in to hydroxymethyl furfural with liberation of water molecule. The obtain furfural reacts with phenol to give complex yellow orange product. The intensity of color develops is directly proportional to amount of hydroxymethylfurfural liberated. Hence the optical density measures of furfural gives amount of glucose present in haemolymph of crab.

19. Comparative Animal Physiology/M.Sc. Zoo-I/Modern College G.K page 19 Reaction of glucose with sulphuric acid PROCEDURE 1. Take a live crab and extract about 2 ml of haemolymph from chelicera. 2. Add it about 8 ml of 5%Ntca solution in to haemolymph and mix thoroughly. 3. The solution is centrifuge for 2 min @ 5oooo rpm at 4oc. 4. Meanwhile ablate both the eyestalk of crab with scissor, care should be taken 5. After 1-2 hours again extract 2ml of haemolymph from crab whose eyes were ablated. 6. Add 8ml 5% TCA solution and centrifuge @ 5ooo rpm for 2min. 7. The supernatant of both the samples were taken and additions were made accordingly as per observation table. 8. A standard glucose solution was used to obtain standard graph. 9. Absorbance of haemolymph sample at 47o nanometer was recorded. 10. The glucose content of haemolymph was calculated before and after eye ablation by using both graphical and calculation method.

20. Comparative Animal Physiology/M.Sc. Zoo-I/Modern College G.K page 20 Result: A: Amount of glucose BEFORE eye stalk ablation by graph: Amount of glucose BEFORE eye stalk ablation by calculation: B: Amount of glucose AFTER eye stalk ablation by graph: B Amount of glucose AFTER eye stalk ablation by calculation OBSERVATION TABLE: SR.NO Std. glucose ml Conc of glucose Dist. Water ml 5% Phenol ml Conc. H2So4 ml O.D .@ 47O nm 1 OO 1.O 1 3 I 2 O.2 O.8 1 3 N 3 O.4 O.6 1 3 C 4 O.6 O.4 1 3 U 5 O.8 O.2 1 3 B 6 1.O OO 1 3 Tion@R.T 3O min

21. Comparative Animal Physiology/M.Sc. Zoo-I/Modern College G.K page 21 Unknown A- before O.4 O.6 1 3 B-After O.4 O.6 1 3 CALCULATION:

22. Comparative Animal Physiology/M.Sc. Zoo-I/Modern College G.K page 22 RESULTS: The crustacean eyestalk is the source of one or more hormones which control blood glucose levels. One of these hormones called the hyperglycemic hormone (HGC, diabetogenic hormone), is able to elevate the level of blood sugar, resembling thus glucagon in vertebrates. Abramowitz et al. (1944) for the first time reported the relation between the eyestalk principle and carbohydrate metabolism in crustaceans. They identified a definite structure in the eyestalk of blue crab Callinectes sapidus which was reponsible for the elaboration of a diabetogenic factor. Rangnekar et al. (1961) found that the ablation of eyestalks led to rise in the blood sugar level of Paratelphusa jacquemontii and injection of eyestalk extract into both destalked and intact crabs produced hypoglycemia, suggesting the presence of hypoglycemic factor in the eyestalks. On The contrary Menon and Sivadas (1967) reported in Scylla serrate that the eyestalks has a hyperglycemic factor and removal of it resulted in hypoglycemia. From these evidences it is clear that crustacean eyestalk has a hyperglycemic hormone which controls the blood sugar level. So experiment can be done in the laboratory on crabs or prawns to see the effect of eyestalk ablation on blood sugar level and to interpret the presence of hypo- or hyperglycemic hormones in eyestalks. REFERENCES: 1. ABRAMOWITZ, A. A., F. L. HISAW AND D. N. PAPANDREA 1944. .The occurrence 2. of diabetogenic factor in the eyestalks of crustaceans. Biol. Bull.,Wood'sHole, 86: 1-5. 3. NELSON, D. H. 1944. A photometric adaptation of the Somogyi's method for the determination of glucose. /. Biol. Chem., 153: 373-380. 4. MENON, K. R. AND P. SIVADAS 1967. Blood sugar regulation in the crab Scylla serrata, effect of injection of eyestalk extract. J. Exp. Biol., 5: 176-178. 5. RANQNEKAR, P. V., P. B. SABNIS AND H. B. NIRMAL 1961. The occurrence of hypoglycemic factor in the eyestalks of freshwater crab Paratelphusa jacquesmontii. J. Anim. Morphol. Physiol, 8: 137-144. 6. ROE, J. H. 1955. The determination of sugar in blood and spinal fluid with anthrone reagent. /. Biol. Chem., 212: 335-343.

23. Comparative Animal Physiology/M.Sc. Zoo-I/Modern College G.K page 23 Date: Name of the student Practical No: Estimation of sugar in rat/crab/human blood. Signature of Practical in charge: Dr.Ravindra Kshirsagar Practical no:

24. Comparative Animal Physiology/M.Sc. Zoo-I/Modern College G.K page 24 Estimation of sugar in rat/crab/human blood. Principle: Sugar/glucose can be estimated by acid phenol and anthrone method. In presence of strong acid glucose gets converted in to hydroxymethyl furfural with liberation of water molecule. The obtain furfural reacts with phenol to give complex yellow orange product. The intensity of color develops is directly proportional to amount of hydroxymethylfurfural liberated. Hence the optical density measures of furfural gives amount of glucose present in haemolymph of crab. Reaction of glucose with sulphuric acid Chemicals: Crab Haemolymph/rat blood/Human blood, Phenol,Sulphuric acid,TCA, Std.glucose distilled Water. Apparatus; Spectrophotometer/colorimeter/centrifuge/testtubes PROCEDURE 1. Take a live crab and extract about 2 ml of haemolymph from chelicera. 2. Add it about 8 ml of 5%Ntca solution in to haemolymph and mix thoroughly. 3. The solution is centrifuge for 2 min @ 5ooo rpm at 4oc.

25. Comparative Animal Physiology/M.Sc. Zoo-I/Modern College G.K page 25 The supernatant of the samples were taken and additions were made accordingly as per observation table. 4. A standard glucose solution was used to obtain standard graph. 5. Absorbance of haemolymph sample at 47O nanometer was recorded. 6. The glucose content of haemolymph was calculated by using both graphical and calculation method. SR.NO Std. glucose ml Conc of glucose Dist. Water ml 5% Phenol ml Conc. H2So4 ml O.D .@ 47O nm 1 OO 1.O 1 3 I 2 O.2 O.8 1 3 N 3 O.4 O.6 1 3 C 4 O.6 O.4 1 3 U 5 O.8 O.2 1 3 B 6 1.O OO 1 3 Tion@R.T 3O min Unknown A- O.4 O.6 1 3 B- O.4 O.6 1 3

26. Comparative Animal Physiology/M.Sc. Zoo-I/Modern College G.K page 26 Calculations: Date:

27. Comparative Animal Physiology/M.Sc. Zoo-I/Modern College G.K page 27 Name of the student Aim: To determine bleeding time and clotting time of human blood. Signature of Practical in charge: Dr.Ravindra Kshirsagar Aim: To determine bleeding time and clotting time of human blood.

28. Comparative Animal Physiology/M.Sc. Zoo-I/Modern College G.K page 28 Principle- Homeostasis is a process of maintaining the blood wall within a closed vascular system. It is a sequence of responses that stop bleeding when the blood vessels are damaged or ruptured. It must be quick and controlled in vision of damage. Three sequential process are observed in case of human blood coagulation – 1. Muscular spasm –Damaged blood vessel cause the circular arranged smooth muscle to contract immediately in a response reaction known as the muscular sparm or vaso contraction these reduces the blood loss for several minutes to hours. During this time the other haemostasis mechanism of rates muscular spasm is activated by the substance released from activated platelets as well as a result of nerve reflexes due to pain. 2. Platelets plug formation- This mechanism is further divided into three steps- a) Platelets Adhesion- Platelets stick to the damaged blood vessel, collagen fiber of connective tissue. b) Platelet relax reaction- Due to the adhesion the platelets gets activated chain an extent projections that unable them to contact, interact and liberate vesicle. These vesicle release ADP, secotonin, thromboxane,A2. These molecule brings vasocontraction. c) Plug formation- Release and ADP molecules make other platelets in vionity.Other platelet in other area allow them to adhere with the activated platelets resulting with the resulting in the formation of platelet plug which effectively inhibit blood loss from small blood vessel damaged.

29. Comparative Animal Physiology/M.Sc. Zoo-I/Modern College G.K page 29 3. Blood clotting- It is a complex cascade of the enzymatic reaction in which each clotting factor activates one or many subsequent molecules in a fixed sequence till the formation of large quantity insoluble protein fibrin factor includes caᶧᶧ and several other inactivated factor synthesized by haepatocyte and release into blood stream. Other various molecules released by the blood and the blood cells and are also known as clotting intrensive or extrensive. However both the pathway lead to the formation of prothombinase. Material required- Pricking needle/ lancet, capillary tube, blotting paper, 70% alcohol or spirit, stop watch, etc. Procedure- Bleeding time: 1. Sterilize the one of the finger and needle as well with the methylated spirit or 70% alcohol. 2. Allow the alcohol to evaporate and prick the tip with the sterilized needle. 3. As soon as bleeding started for recording of time stop watch was switched on. 4. Filter paper strip was taken and its ventricle surface end was touched for observing blood, precaution should be taken to avoid touching of filter paper with finger tip. 5. When the bleeding stop time was recorded, this was bleeding time. 6. The procedure was repeated thrice. 7. An average value of three reading was taken. Clotting time- 1. One of the fingers was swabbed with methylated spirit or 70% alcohol. 2. Alcohol was allowed to evaporate from the finger tip. 3. It was pricked with the sterilized needle. 4. As soon as bleeding start stopwatch started for recording the time.

30. Comparative Animal Physiology/M.Sc. Zoo-I/Modern College G.K page 30 5. First drop of the blood was wiped out with the filter paper and the fingertip was pressed for releasing fresh blood which was collected in a capillary tube up to 2/3 of its length. 6. After ever 30sec 1cm of capillary tube were broken to check whether the fibrin string had formed or not. 7. As soon as fibrin string appeared stop watch was stopped and time was recorded. This is a way the clotting time. Results- Bleeding time- Clotting time- Conclusion- Normal bleeding time of human varies from 2-5 min. This bleeding time may change in abnormal condition which can lead to severe hemorrhage condition. It could be possible either due to the factors of essential clotting factor or due to some disease which prohibit the production of these molecules in adequate quantity. Normal clotting time in human varies from 5-10min. variation of clotting time indicates abnormalities in clot factor lX (ca++ ions). Deficiency of ca++ ions increases CT and vice versa.

31. Comparative Animal Physiology/M.Sc. Zoo-I/Modern College G.K page 31 Date: Name of the student Practical: Body size and oxygen consumption in aquatic animals (crab/fish). Signature of Practical in charge: Dr.Ravindra Kshirsagar

32. Comparative Animal Physiology/M.Sc. Zoo-I/Modern College G.K page 32 Aim: Body size and oxygen consumption in aquatic animals (crab/fish). Principle – O₂ is one of the most important elements for living system as it is essential for all the cells to carry out its fuel combustion process aerobic respiration. Due to this reaction, O₂ is often termed as biological life support and medicine. Terrestrial animals easily obtained O₂ for their rise directly from the surrounding environment atmosphere, where it is adequately available. However in aquatic ecosystem animals are dependent on dissolved O₂ in H₂O. The dissolved oxygen contain of H₂O results from – 1. The photosynthetic and respiration activities of the biota in the open H₂O, the benthos and the autotrophs. 2. The diffusion gradient of the air H₂O interfere and distribution by wind and wave thriven mixing. 3. The insufficient O₂ in H₂O bodies leads to aerobic condition and start releasing pungent odor and toxic gases such as H₂O. Hence measurements do in H₂O influence profile of aquatic life. In sufficient supply of O₂ conditions following reactions takes place – O₂ + C CO₂ ↑ O₂ + S SO₄⁻ O₂ + P PO₄⁻ O₂ + N NH₃↑ Dissolved oxygen can be determined either by the Wrinkler method or Iodonutric filtration. The working principle based on the fact the equivalent amount of iodine liberated is directly proportional to O₂ present in H₂O sample. Manganese white precipitate of manganese hydroxide which is presence of O₂ gets converted into free brown coloured compound.

33. Comparative Animal Physiology/M.Sc. Zoo-I/Modern College G.K page 33 In strong acid medium, manganese ion are reduced due to presence of iodine. Iodine ions gets converted into free iodine atoms which is equivalent to the original concentration of dissolved oxygen in the H₂O. free iodine ions can be liberated against hypo solution of Na₂H₂O₃ using starch as a indicator. Reactions – MnSO₄ + 2NaOH Mn(OH)₂ + Na₂SO₄ 2Mn(OH)2 + O2 2 Mn (OH)2 (( white ppt) Mno(OH)2 + 2H2SO4 + 2NaI MnSO4 + Na2SO4 + 3H2O + I2 In titration I2 reacts with Na2S2O3, Na2S2O3 + I2 Na2S4O6 + NaI Material Required: Biological- Crab / Fish Chemicals- Manganese sulphate, NaOH, NaI2, Conc. H2So4, Na2S2O3, Potassium hydroxide, Filtered pond water Reagents: 1) MnSO4 solution Dissolve 480gm of MnSO4.4H2O in 1 litre of DW or Dissolve 400gm of MnSO4. 2 H2O in 1 litre of DW or Dissolve 364 gm of MnSO4.2 H2O in 1 liter of DW. 2) Alkaline Iodide: Dissolve 500gm of NaOH and 135gm of NaI2 in DW and make up the volume upto 1 litre or

34. Comparative Animal Physiology/M.Sc. Zoo-I/Modern College G.K page 34 Dissolve 700 gm of KOH and 150 gm of KI in DW and dilute upto 1 liter 3) Starch Solution: Prepare an emulsion of 6 gm starch in small beaker in DW. Pour this emulsion of 750 ml DW and make up the volume upto 1 liter. Boil the solution till it gets transparent and allow it to cool down. The clear supernatant is used as starch indicator it can be preserved for long time by adding 1.5 gm per liter salicylic acid. 4) Sodium thiosulphate stock solution (1N): dissolve 248.2 gm of Na2S2O3 H2O in boiled and cooled DW and make volume upto the 1 liter. This solution can be preserved by adding 5 ml chloroform/ liter or dissolving 1 gm NaOH / liter. 5) Standard working solution of Na2S2O3 (0.025 N ): a. Dilute 25 ml of stock solution (1 N ) og Na2s3O3 to 1000 ml with DW. b. Dissolve 6.205 gm og Na2S2O3 in freshly boiled and cooled DW and make the volume upto 1 liter. This solution can be preserved by dissolving 5 ml chloroform / liter or dissolving 0.4 gm NaOH / liter. 6) conc. H2SO4 PROCEDURE : Fixation of dissolved O2 : 1. The fixation was done immediately after the collection of sample from the field ( delay in the fixation may lead to error following steps were carried out to fix the DO. 2. H2O sample was taken in 250 ml / 300 ml volume of the sample bottle. 3. Stopwatch removed from the sample bottle and 1 ml manganese sulphate solution was added by inserting the tip of glass pippete just below the H2O surface.

35. Comparative Animal Physiology/M.Sc. Zoo-I/Modern College G.K page 35 4. 1 ml of alkaline iodide solution was also added in the similar manner. 5. Stopper of sample bottle was replaced and H2O sample was mixed properly by the bottle severed time. 6. Precipitate from were allowed to settle down for a few min. 7. 1 ml conc, H2SO4 was added slowly to the wall of the sample bottle. 8. Sample bottle was inverted several time to mix it properly. 9. The sample bottle was left to stand without any disturbance for atleast 10 – 15 min. TITRATION : 1. Took 50/100 ml of H2O sample in which DO had been fixed in a conical flask. 2. This sample was immediately filtrated with 0.025 N Na2S2O3 solution. 3. As soon as the solution turned pale yellow in color 1 ml of indicator solution (starch solution) was added immediately. The solution color changed from pale yellow to blue. 4. Titration was continued slowly until the first disappearance of blue color. 5. The used volume of Na2S2O3 solutions were recorded in ml. 6. The titration was repeated thrice to calculate the DO.

36. Comparative Animal Physiology/M.Sc. Zoo-I/Modern College G.K page 36 FORMULA : O2 / Lit ( mg ) = used volume of titrant in ml × 1000 × 0.2 Volume of H2O in ml Calculation: 1 ml of 0.025 N sodium sulphate is equivalent to 0.2 mg of dissolved oxygen. OXYGEN CONSUMPTION BY CRAB :  Initially water sample was collected before putting the crab in the oxygen chamber.  O2 fixation and filtration steps were carried out to calculate amount of dissolved oxygen in the water sample.  Crab was introduced into the O2 chamber and chamber was sealed by the water throw glass pipe.  The set up was left undisturbed for 1 hr, so that crab can consume O2 from the given water source only.  After an hour only outlet tube has been opened to collect water sample.  Again O2 fixation and filtration were repeated to calculate the amount of dissolve oxygen remaining.  Differences were calculated to know the O2 consumed by the animal. End point blue to colorless

37. Comparative Animal Physiology/M.Sc. Zoo-I/Modern College G.K page 37 Date: Name of the student Aim: Capillary circulation in the foot-web of frog/tail-fin of fish Signature of Practical in charge: Dr.Ravindra Kshirsagar

38. Comparative Animal Physiology/M.Sc. Zoo-I/Modern College G.K page 38 Aim: Capillary circulation in the foot-web of frog/tail-fin of fish Purpose: To see how capillaries appear and work in a living organism. Materials: cotton petri dish 2 glass slides Goldfish eyedropper pipette Microscope beaker Procedure: 1. Gather your materials for your station, including your fish in a beaker. 2. Soak a piece of cotton (enough to wrap around the fish like a blanket) in the water in the beaker with the fish. 3. Flatten out (unwrap) the piece of soaking wet cotton ball and place it in the bottom of a clean petri dish. 4. Gently remove the goldfish with a clean hand from your beaker, and place it GENTLY onto the wet cotton so that the gill and head areas (not mouth!) are covered and the tail fin, the caudel fin, is showing.

39. Comparative Animal Physiology/M.Sc. Zoo-I/Modern College G.K page 39 5. Place the petri dish on the stage of the microscope so that the tail fin is visible under the low power objective. Examine the goldfish’s tail under low power only. Move the petri dish around until you see blood moving in the blood vessels. 6. While one-partner views the caudal fin under the microscope, the other partner should be monitoring the Fish’s wellbeing. Using the eyedropper, count, “1, 2, 3, 4, 5…..drip” and place a drop of water on to the cotton ball above the fish’s gills. This constant adding should help the fish breathe while out of water. 7. Locate a blood vessel in which blood cells are passing in a single file. This is a capillary. Note the direction of the flow of blood. Follow the capillary in the direction opposite the blood flow to where it joins a slight larger vessel (arteriole). Then follow it in the direction of blood flow until it joins a slighter larger vessel (venule). In your lab notebook, draw and label the different types of blood vessels that you see. U arrows to show the direction of the blood flow. 8. If at any time while viewing the capillaries the blood appears to dramatically slow down, return the fish to the beaker of water to avoid trauma. When finished, clean up your station and return the goldfish alive and well! Tail-fin of fish

40. Comparative Animal Physiology/M.Sc. Zoo-I/Modern College G.K page 40 The most important process associated with the circulatory system is the exchange of substances between the blood and the interstitial fluid. The collection of vessels that is involved in this exchange is often referred to as the MICROCIRCULATION and consists of arterioles, met-arterioles, capillaries and venules. A conceptual diagram of a typical capillary bed is shown below. AIM: ESTIMATION OF CHLORIDE CONTENT OF CRAB. Principle: Chloride is one of the essential and important minerals for various body function. It provides 2/3 of plasma and anions of the body. Its chief factor is regulatory body temperature reaction..Chloride like NaCl, KCL are important agents in regulation of osmotic pressure in body. HCL of gastric juice derived from blood. Chloride shift is an important phenomenon by which ions shift from plasma to cell and helps carrying CO2 and regulation of blood reaction equilibrium usually of altering of NaCl. It is mainly stored in the form of chloride under skin and substaintial tissue. As Cl¯ in blood are not free and are always combined with Na˖. Any cl¯ estimation by any method is impossible. Hence , we determine the iodine content of the blood sample, taken in first

41. Comparative Animal Physiology/M.Sc. Zoo-I/Modern College G.K page 41 deproteinized to remove protein and filtrate is treated with AgIO3 (silver iodate). The filtrate contain NaCl after adding AgIO3 , silver chloride precipitate and instead of Cl, iodine ion get attached to Na++. Hence, Cl¯ and iodine replacement takes place. Now the titration has NaIO3 which is treated with KI. Iodine is liberated using starch has indicator. This liberated iodine using starch is estimated and treated against sodium thiosulphate. This reaction takes place as : NaCL + AgNO3 NaIO3 + AgCl NaIO3 + SKI + H3PO4 3Na + 2HPO4 + 3I2 + 3H2O 3I2 + 2Na2SO2 + O3 2NaI + Na2S4O6 Requirement : Blood sample, 1 % phosphotungstic acid, saturated KI, 0. 025 NNa2SO3 and starch indicator , wattmanns filter paper. PROCEDURE :

42. Comparative Animal Physiology/M.Sc. Zoo-I/Modern College G.K page 42 1. Take 25 ml of 1 % phosphotungstic acid. 2. Add 1 ml of haemolymph. 3. Filter with wattmanns filter paper. 4. Filtrate + add 40 mg AgNO3. 5. Again filter after adding AgNO3. 6. Take 10 ml of filtrate. 7. Add 1 ml saturated KI. 8. Titrate against sodiuym thiosulphate(0. 025 NNa2SO3). 9. Amber color develops + add starch indicator. 10. Titrate till color changes from blue to colorless. 11. Note the burrette reading. RESULTS:

43. Comparative Animal Physiology/M.Sc. Zoo-I/Modern College G.K page 43 TO STUDY THE EFFECT OF INSULIN ON BLOOD SUGAR LEVEL OF MICE PRINCIPLE: Insulin is hormone secreted by B-cells of islets of Langerhans of pancreas. Insulin maintain sugar level in blood by promoting glycogenesis and B- OXIDATION in pathological condition diabetes mellitus the blood sugar level of patient elevates than the normal range hence to maintain the normal blood sugar level either insulin tablets or commonly insulin injections are prescribed by the physicians. Chemicals required: Biological Material: Mice Chemicals: Ether/chloroform, 5%TCA,5%Phenol,Sodium citrate or any anticonticoagulant, Insulin 4o units/ml, Standard glucose solution 1oo ug/ml Equipments: spectrophotometre, glass ware Procedure: 1. The mouse was anesthesized either by using ether or chloroform. 2. About 1 ml of blood was collected in sodium citrate tube. 3. Take o.5 ml of blood add 1.5ml 5% TCA Solution. 4. The sample was centrifuge at 4OOO rpm for 5 min@ 4oc. 5. The obtained supernatant was separated and used as sample solution. 6. In second part: 1 ml insulin was injected intraperitoneally to mouse. 7. The mouse was left in normal condition for half an hour. 8. Again about I ml of blood was collected from the same mouse 9. Same procedure as above was repeated to obtain second set of blood sample. 10. Additions were made in both the blood samples as well as for standard glucose according to observation table. 11. Optical density of each sample was recorded @ 47O nm.

44. Comparative Animal Physiology/M.Sc. Zoo-I/Modern College G.K page 44 12. A graph was plotted for standard solution of glucose and concentration of blood sample calculated with the help of it. OBSERVATION TABLE: SR.NO Std. glucose ml Conc of glucose Dist. Water ml 5% Phenol ml Conc. H2So4 ml O.D .@ 47O nm 1 OO 1.O 1 3 I 2 O.2 O.8 1 3 N 3 O.4 O.6 1 3 C 4 O.6 O.4 1 3 U 5 O.8 O.2 1 3 B 6 1.O OO 1 3 Tion@R.T 3O min Unknown A- before O.4 O.6 1 3 B-After O.4 O.6 1 3 CALCULATION: Calculations:

45. Comparative Animal Physiology/M.Sc. Zoo-I/Modern College G.K page 45 DETERMINATION OF URIC ACID CONCENTRATION IN SERUM Principle. Reduction of uric acid with phospho wolframic acid (the part of Folin’s reagent) forms coloured compounds, which can be measured using photoelectrocolorimetry. Procedure. Add 1.5 ml of serum, 1.5 ml of distilled water and 1.5 ml of 20% trichloroacetic acid (CCl3COOH) into the centrifuge tube. Mix the contents of the tube thoroughly. After 30 minutes centrifuge the tube at 3000 rpm. Then add into two empty tubes the following reagents: Sample Standard Supernatant CCl3COOH Distilled H2O Na2CO3 Folin’s reagent. (ST) .5 ml 0.5 ml 0.5 ml 0.7 ml 1 drop (S) 1.5 ml 0.7 ml 1 drop After 10 minutes perform a calorimetric measurement, using a green light filter. Uric acid concentration is calculated using the formula: Cs (mg/1 ml) *59 = Cs (μmol/l), here Cs concentration of uric acid in the serum (mg/dl); CST concentration of uric acid in the standard (I) solution (0.02 mg/ml); Es extinction of the sample (II); EST – extinction of the standard solution; a volume of the supernatant. 59 – the convertion coefficient from mg/1 ml to μmol/l.

46. Comparative Animal Physiology/M.Sc. Zoo-I/Modern College G.K page 46 Principles Of Colorimetry Colorimetry is the techniques that is frequently used in biochemical investigations. This involves the quantitative estimation of colors. This means that if you want to measure the quantity of a substance in a mixture, you could use the technique of colorimetry, by allowing the substance to bind with color forming chromogens. The difference in color results in the difference in the absorption of light, which is made use of here in this technique called colorimetry. Apparatus: The instrument use for colorimetry is colorimeter. This appartus will comprise of the following parts: 1. light source 2. filter (the device that selects the desired wavelenght) 3. cuvette chamber (the transmitted light passes through compartment wherein the solution containing the colored solution are kept in cuvette, made of glass or disposable plastic) 4. detector (this is a photosensitive element that converts light into electrical signals) 5. Galvanometer (measures electrical signal quantitatively) The spectrophotometer also works on a similar principle. Beer-Lambert’s Laws:  Beer’s Law According to Beer’s law when monochromatic light passes through the colored solution, the amount of light transmitted decreases exponentially with increase in concentration of the colored substance. It = Io e-KC  Lambert’s Law According to Lambert’s law the amount of light transmitted decreases exponentially with increase in thickness of the colored solution. It = Io e-kt Therefore, together Beer-Lambert’s law is: IE/Io = e-KCT

47. Comparative Animal Physiology/M.Sc. Zoo-I/Modern College G.K page 47 where, IE = intensity of emerging light Io = intensity of incident light e = base of neutral logarithm K = a constant C = concentration T = thickness of the solution Steps for operating the photoelectric colorimeter: 1. Choose the glass filter recommended (see table below) in the procedure and insert in the filter. 2. Fill two of the cuvette with blank solution to about three-fourth and place it in the cuvette slot. 3. Switch on the instrument and allow it to warm up for 4 – 5 minutes. 4. Adjust to zero optical density. 5. Take the test solution i another cuvette and read the optical density. 6. Take the standard solution in varying concentration and note down the optical density as S1, S2, S3, S4, S5 and so on. 7. A graph is plotted taking concentration of standard solution versus the optical density. 8. From the graph the concentration of the test solution or the unknown solution can be calculated.

48. Comparative Animal Physiology/M.Sc. Zoo-I/Modern College G.K page 48 Table for choosing the wavelength of absorption:

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