DETAILED RESEARCH & DEVELOPMENT PROJECT PROPOSAL
(For the Component Project)
(To be accomplished by the researcher)
(1) Title/Leader/Gender/Agency/Address/Telephone/fax/E-mail
Program Title:
Animal Physiology Laboratory Special Project
Project Title:
Comparative Analysis of Blood Characteristics of Different Animals in Relation to Respiration
Leader/Gender: Nikki Vanesa Tampon/Female
Jasmin E. Alojado/Female
Ysabela Marie C. Rabaya/Female
Stephen Adrian Dayap/Male
Agency/Address:
Department of Biological and Environmental Science College of Science and Mathematics
University of The Philippines Mindanao Campus, Mintal, Tugbok District, Davao City
Telephone/Fax/E-mail: N.V. Tampon: 09238374947; nttampon@up.edu.ph
J.E. Alojado: 09655006308; jealojado@up.edu.ph
Y.C. Rabaya: 09274650413; ysabelarabaya@gmail.com
S.A. Dayap: 0907 855-3006; hernan.adrian12@gmail.com
(2) Cooperating Agencies
University of the Philippines Mindanao
College of Science and Mathematics
(3) Research & Development Station
Department of Biological and Environmental
Science College of Science and Mathematics
University of The Philippines Mindanao
Campus
(4) Site of implementation/Municipality/District/Province/Region
Davao City, Region 11, Philippines
Research: Development:
____ Basic ___X__ Pilot Testing ___X___ Single Agency
__X__ Applied _____Tech. Promotion/
______ Multi Agency
Commercialization
(7) Sector/Commodity (8) Discipline
Physiology

(9) Significance
There are many different species of animals present in the Philippines, it being one of the hotspots for species diversity. Aside from the taxonomic characterization that could be used to identify such species, blood cell count and morphology can also be used for differentiation. Blood cell morphology and its amount in an animal could also be used to assess the respiration capacity of the specific animal, which could imply the mechanism in which it respires, thus it could also be used as a basis on identifying the type of environment it lives in. Knowing this correlation, we could then use these evidences to assess the certain environmental adaptations made by these species in order to survive in its highly dynamic and fastly changing habitat. We can then utilize this knowledge in conservation and protection of these animals as well as in the field of preserving the habitats of these animals.
(9-A) Project Description
The proposed study is targeted to give another perspective in species identification through respiratory capacities and assessment of environmental conditions in which these species lives in by means of blood cell count and morphology. The proper way of extraction, accurate cell counting, and keen observation of morphology of the the blood samples is vital in reaching a conclusive and reliable result of this study.
(10) Objectives
The proposed study is aimed to:
1. Extract and collect blood samples from different species of animals;
2. Perform blood cell counting and blood cell morphology assessment on the blood samples gathered;
3. Make correlations on the blood cell count and morphology assessment done and the respiratory capacity it would indicate;
4. Connect all these correlations with the kind and type of environment in which these animals live in.
(11) Review of Literature
Blood characteristics of animals varies from one species to another. In correlation to the evolution of animals, most animals have nucleated red blood cells while for mammals, the nucleus of their red blood cells degenerate as the cells become mature (Fox, 2003). Evolution of animals caused the differences in blood cell characteristics that reflects their way of living, habitat and niche.
BIRDS
One of the unique characteristics of birds are to be able to fly in high altitude without becoming comatose unlike other mammals (Tucker 1968). According to Kahle and Becker (1999) this is because avian blood has a significantly lower oxygen affinity compared to other animals with PsO values typically ranging from 40 to 50 mm Hg. The low affinity of the avian blood to oxygen is an advantage to them during a flight, since this enables a more efficient oxygen unloading during respiration (Benesch and Benesch, 1969).
FISHES
Interest in understanding the blood cells of fish dates back to the mid 1800’s. Fishes live in bodies of water in which they respire through their gills. The exchange of dissolved oxygen in water and carbon dioxide is mainly done by the transport of blood (Collins et. al., 2016). According to Hughes and
Shelton (1962), the effectiveness of transfer of these gases depended on the capacity rate ratio of blood and water and the conditions for transfer across the gills, as well as the countercurrent flow of blood and water.

Fish erythrocytes resemble the avian/reptilian erythrocytes in which it is described as oval in shape with abundant smooth eosinophilic cytoplasm and a central, oval-shaped condensed nucleus. Fish lymphocytes are small round cells with a high Nucleus:Cytoplasm ratio and a rim of smooth light blue cytoplasm around the large oval-round condensed nucleus. The monocytes, on the other hand are described as large, usually round cells with abundant blue cytoplasm often containing vacuoles (Arnold,
2009).
CATS
Cats have four-chambered hearts such as humans however, their blood morphology is relatively different. Feline erythrocytes display moderate anisocytosis, a condition where the size of the red blood cells have unequal sizes, and lack central pallor which is the pale area at the center of the red blood cell (White and Reine, 2009). Their hemoglobin is also more susceptible to oxidant injury because it contains eight weak oxidizable sulhydryl groups and easily dissociates from tetramers to dimmers
(Harvey, 2017a). This is also why they have Heinz bodies which are inclusions in the RBC that contain denatured hemoglobin (Harvey, 2017a).
One unique characteristic of the feline blood is the presence of Dӧhle bodies which are made up of retained aggregates of the rough ER in the neutrophils of healthy cats. These are inclusions are believed to be an indication of mild toxicity of the cytoplasm (Harvey, 2017b).
MOUSE
Mice, which is a relatively small mammal, have smaller erythrocytes with shorter life span compared to other mammals. However, they maintain a a rather regenerative state with 1% to 6% reticulocytes in their circulation which is relatively higher than in adult humans which only has 0.5% to 1% (Szigeti,
2014). This compensates for the small size and short lifespan of their erythrocytes. They also have very high platelet count compared with other mammals which is a probable cause for platelet clumping
(O’Connell et al., 2015).
FROG
Frogs can live both in water and in land and can respire through their skin. It only have three chambers in its heart, two atria and a single ventricle (Fox, 2003). The liquid portion of frog’s blood contain solid elements such red blood cells and white blood cells (Nikon, 2018). Red blood cells of frogs are nucleated with a cell size of approximately 18x10um (Boehler et al., 1973). Erythrocytes of tadpoles are circular, oval or elliptical in shape with centric nuclei and it size unusually smaller than normal erythrocytes (Hota et al., 2013). Adult frogs have elliptical shaped- red blood cells, nucleated and spindle shaped thrombocytes and numerous lymphocytes (Hota et al., 2013). The size and shape of erythrocyte give an indication of the surface available for the exchange of gases in respiratory functions
(Hartman et al., 1964). According to Hartmann et al. (1964), various studies have suggested that having a elliptical shaped red blood cell than a spherical onen is more efficient because of greater gas exchange because the cell have higher surface area.
(12) Scientific Basis/Theoretical Framework
Erythrocytes are the most important carrier of oxygen and carbon dioxide. Differences in erythrocytic morphology of every animal might be correlated with the mechanism of its respiration and its evolution to adapt well on its designated environment. For instance, elliptical morphology of red blood cells is more efficient in gas exchange compared to spherical or circular erythrocytic shape. Non-nucleated red blood cells in mammals is an adaption to acquire more oxygen in blood and increase circulatory effici ency along with its system’s complexity. Furthermore, amphibians and fishes have different mechanisms of respiration which involve other organs like skin and gills, hence these animals exhibit larger red blood cells in comparison to animals with very efficient respiratory system like birds, which exhibits the smallest size of red blood cells. This study aims to assess thoroughly the correlation with blood morphology along with its cell count and its respiratory mechanism to provide further analysis on

adaptation and evolution of the diversity of species.
(13) Methodology
13.1 Preparation of Blood Smear
Observing blood cell morphology requires the preparation of thin blood smears. Thin layer of blood smeared in a glass slides that is added with a stain enables microscopic examination and comparison of different blood characteristics. The stain that is widely used in staining blood is Giemsa stain. A histological stain which colors the nuclei dark blue and the cytoplasm into blue or pink depending on the acidity of the cytoplasmic contents.
A small drop of blood that is obtained from the samples, will be placed in a clean glass slide. The blood in the slide must only be about 2 mm in diameter. The slide will be place in a flat surface securing the slide on one hand, while the other hand will be holding a second slide at a 30 degrees angle from the slide with the blood. The blood will be draw back against the second slide hence allowing the blood to spread almost to the edges of the slide. In one swift move, the second slide will be pushed forward and thin film of blood with feathered edges will remain on the slide. Make sure that the blood film will be airdried completely before subjecting the slide to Giemsa Stain. The slide will be labeled according to its designated samples.
13.2 Microscopy
The thin blood smears will be observed under 400x to 1000x and the different morphological characteristics of the cell will be noted.
13.3 Blood Counting
The RBC diluting pipette will be filled with sample blood up to 0.5 mark. The blood will be diluted using
Grower’s or Hayem’s fluid up to the 11th mark. The tip of the pipette will be secured using a finger and the rubber tubing will be removed on the other end and will be secured by the thumb. The pipette will be shaken vigorously in horizontal motion. The coverslip will be placed at the middle of a hemocytometer. The tip of the pipette will be placed at the edge of the cover slip and enough amount of diluted blood will be run under the slip. The slide will be examined under the microscope and count the red blood cells located inside the four corners and center tertiary squares. Only the red blood cells touching the upper and right margins will be considered. The total amount of red blood cells will be counted and will be multiplied by 10, 000.
(14) Major Activities/Work plan (Enumerate in chronological order the task to be undertaken. Use gantt chart.
See Form 2B-1)

(15) Expected Output
15.1 Blood Cell Count and Morphology of Sampled Animals
15.2 Respiratory Capacities of Sample Animals based on Blood Cell Count and Morphology
15.3 Establishment of correlations between blood cell count and morphology and respiratory capacity of sampled animals
15.4 Linking patterns of blood cell count and morphology, respiratory capacity and environmental conditions of the sampled animals
(16) Targets Beneficiaries
The target beneficiaries of the proposed study in the academe are the conservation and environmental biologists and systematists. This study would help in the analyses of the correlation between blood cell count and morphology, respiratory capacity, and environmental conditions in which animals live in. Knowing these linkages, one can have insights on the mechanisms and adaptations made by the sampled animals in order to survive in its habitat. This study would also broaden the knowledge of the scientific community in assessing respiratory capacities and correlating it with environmental factors present.
(17) Personnel Requirement
Percent time devoted to the
Project
Program/Project Leader (1): 40%
University Research Associates (3): 60%
(18) Budget (See form 2B-2)
(19) Literature Cited
Arnold, A. 2009. Hematology of Fish: WBC and RBC Cell Morphology. Proceedings of the
ACVP/ASVCP Annual Meetings 1-3.
Benesch, R. and R. E. Benesch. 1969. Intracellular organic phosphates as regulators of oxygen release by haemoglobin. Nature 221: 618-622
Boehler R.W., Catanzoro A.J., Stein, J. H., Hunter, W. (1973) The Radiolabeled Frog
Red Blood Cell. Retrieved from https://www.ahajournals.org/doi/pdf/10.1161/01.RES.32.6.718
Collins, S., Dornburg, A., Flores, J., Dombrowski, D., Lewbart, G. 2016. A comparison of blood gases, biochemistry, and hematology to ecomorphology in a health assessment of pinfish ( Lagodon rhomboides) . PeerJ . 2016; 4: e2262.
Fox, S.I., 2003. Human Physiology, 8th ed, Mcgraw-Hill.
Harvey, J.W., 2017a. The feline blood film: 1. Techniques and erythrocyte morphology. J. Feline Med.
Surg. 19, 529 –540.
Hartman, F. A. and Lessler, M. A. (1964) “Erythrocyte measurements in fishes amphibians and reptiles,” The Biological Bulletin, vol. 126, no. 1, pp. 83–88,
Harvey, J.W., 2017b. The feline blood film: 2. Leukocyte and platelet morphology. J. Feline Med. Surg.
19, 747 –757.
Hota, J., Das, M. and Mahapatra, P. K. (2013), “Blood Cell Profile of the Developing Tadpoles and
Adults of the Ornate Frog, Microhyla ornata (Anura: Microhylidae),” International Journal of Zoology, vol. 2013, Article ID 716183, 14 pages. https://doi.org/10.1155/2013/716183.
Hughes, G., Shelton, G. 1962. Respiratory mechanisms and their nervous control in fish. Adv.

Comp. Physiol. Biochem. 1, 275-364.
Kahle, S. and Becker, P.H., 1999. Bird blood as bioindicator for mercury in the environment.
Chemosphere , 39 (14), pp.2451-2457.
Nikon (2018) “Frog Red Blood Cells” Retrieved from https://www.microscopyu.com/gallery-images/frogblood-cells
O’Connell, K.E., Mikkola, A.M., Stepanek, A.M., Vernet, A., Hall, C.D., Sun, C.C., Yildirim, E., Staropoli,
J.F., Lee, J.T., Brown, D.E., 2015. Practical murine hematopathology: a comparative review and implications for research. Comp. Med. 65, 96 –113.
Szigeti, R.G. 2014. Reticulocyte Count and Reticulocyte Hemoglobin Content [WWW Document]
Medscape URL https://emedicine.medscape.com/article/2086146-overview (accessed 12.3.18)
Tucker, V. A. 1968. Respiratory physiology of house sparrows in relation to high-altitude flight. J.
Exp.Bid. 48 : 55-56
White, C., Reine, N., 2009. Feline Nonregenerative Anemia : At a Glance 1–7.