A simple and rapid method for blood collection from walking catfish, Clarias batrachus (Linneaus, 1758)

Item Type article

Authors Argungu, L.A.; Siraj, S.S.; Christianus, A.; Amin, M.S.N.; Daud, S.K.; Abubakar, M.S.; Abubakar, I.A.; Aliyu-Paiko, M.

Download date 01/10/2021 02:13:44

Link to Item http://hdl.handle.net/1834/37860 Iranian Journal of Fisheries Sciences 16(3) 935-944 2017

A simple and rapid method for blood collection from walking catfish, Clarias batrachus (Linneaus, 1758)

Argungu L.A.¹; Siraj S.S.¹; Christianus A.1,4*; Amin M.S.N.1; Daud S.K.²; Abubakar M.S.3; Abubakar I.A. 3 ; Aliyu-Paiko M.4

Received: July 2015 Accepted: April 2016

Abstract Blood is collected from experimental animals for a wide range of scientific purposes including; hematology, clinical biochemistry parameters, immunology, studies in bacteriology, parasitology and investigations in reproductive performance and health. The number of methods employed to collect blood from fish include; the puncture of caudal vein, dorsal aorta or cardiac vessels and the severance of the caudal vein. Unfortunately, all these procedures are practically found to be slow and stressful to Clarias batrachus, including the popular caudal vein approach, likely due to the small size of caudal veins relative to the size of the species. In line with the universal ethical recommendations for taking blood from small research animals, we propose an alternative one-operator approach for C. batrachus that is simple, rapid and without the need to sacrifice the fish as with other methods. This procedure targets the dorsal aorta (a relatively larger blood vessel) in a sedated fish, punctured by inserting a needle directly from the anterior part of the anal fin about 2-5 mm behind the genital papilla, to Downloaded from jifro.ir at 16:46 +0330 on Sunday March 4th 2018 draw the desired amount of blood. The technique is a one-operator procedure not requiring the help of an assistant or any special equipment to restrain the fish. The operation of the protocol is unique since it permits the continuous collection of blood from the same experimental fish over a varied time course and reduces the need for a large number of replicate animals. The advantages of the proposed protocol are also highlighted and discussed in detail.

Keywords: Haematology, Catfish, Aquaculture, Blood sampling, Clarias batrachus

1-Department of Aquaculture, Faculty of Agriculture, Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia 2-Departments of Biology, Faculty of Science, Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia 3-Department of Veterinary Pathology and Microbiology, Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia 4-Institute of Bioscience, Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia *Corresponding author’s Email: [email protected] 936 Argungu et al., A simple and rapid method for blood collection from walking catfish, Clarias …

Introduction available for blood collection from fish, Clarias batrachus is one of the most including the puncture of caudal vein, notable catfishes in Asia (Argungu et dorsal aorta or cardiac vessels, al., 2012) and its popularity has decapitation and the severance of increased significantly as an important caudal vein (MUAWC, 2008). commercial aquaculture species However, the choice of sampling (Zonneveld et al., 1988; Knud-Hansen technique utilized by a researcher et al., 1990; Kumari and Sahoo, 2006; depends on a number of factors such as Sahoo et al., 2007) and for aquarium the health status and size of fish, the trade in recent times among the fish quantity of blood required to be farming communities in Asia (Ng and collected and the fate of the animal Kottelat, 2008). This is better (terminal or non-lethal) in a study appreciated, considering that consumers (Svobodora and Vykusova, 1991). prefer it more, than Clarias gariepinus Overall, the chosen sampling approach in some parts of Asia because of its should be the least stressful, to avoid excellent taste and rough muscle mass. interference with the outcome of the This is in spite of the better growth study. This consideration makes it performance achieved with the latter necessary for the chosen methodology species when both are farmed (Rahman to be refined, in order to also comply et al., 1995). C. batrachus typically with International ethical and attains a standard length of 22.5-30 cm, procedural requirements, such as the although individuals twice this size has Australian Code of Practice for the been reported (Masterson, 2007). Care and Use of Animals for Scientific Lately, research efforts on C. batrachus Purposes (MUAWC, 2008). are focused on its reproductive The most popular technique, Downloaded from jifro.ir at 16:46 +0330 on Sunday March 4th 2018 performance, especially with regards to targeting the caudal vein to draw blood selection of matured candidates for (MUAWC, 2008) is practically slow induced breeding. To carry out such and stressful, particularly when applied studies effectively, the need arises for to C. batrachus. This is likely due to the researchers to collect blood from the small size of caudal veins in the experimental animals, for further species. Furthermore, the other processing (Kori-Slakpere, 1985). conventional methods are very Collection of blood from cumbersome and less convenient to experimental fish is for a wide range of sample blood in the species (via non- scientific purposes including lethal means). This is particularly a hematology, clinical biochemistry challenge in experiments which require parameters, studies in bacteriology, continuous blood collection over a parasitology and investigations in varied time course and need the animal reproductive performance and health to be kept alive. (DFO, 2004). A number of methods are Iranian Journal of Fisheries Sciences 16(3) 2017 937

It is an ethical obligation for researchers of clove oil solutions diluted in 5 L of to ensure that laboratory animals in water), for 4-6 min. Before the blood their care during experimentation are collection procedure, we subjected the not subjected to unnecessary stress fish samples to morphometric (DFO, 2004). Consistent with this measurements, to determine their body therefore, the objective of the present weight, as well as total and standard exposition was to develop a one- lengths. Complete sedation was operator approach that is simple and confirmed when a fish no longer rapid for sampling blood from C. responded to external stimuli, yet slight batrachus, without the need to sacrifice gill movements were still observable. the fish. The advantages of the Blood collection was accomplished proposed protocol are also highlighted as follows; the completely sedated fish and discussed in detail. was removed with the aid of a net from the anesthetic solution and placed on a Materials and methods clean towel on its side. Using the towel, The specimens we used to develop this the fish was carefully lifted to a protocol were C. batrachus brood fish, comfortable handling position with one with an average body weight and total hand, in such a way that the fish was length of 186 g and 28 cm, respectively. upside down and the head away from, The live fish were caught from the wild but tail nearer to the individual carrying by fishermen in Negeri Sembilan, out the procedure. With the other hand, Malaysia where they were purchased a 23G needle was inserted into the fish and transported to University Putra at a point anterior to the anal fin, and Malaysia Aquaculture hatchery unit, about 2-5 mm behind the genital Puchong-Selangor, Malaysia. The fish papilla. The insertion of the needle into Downloaded from jifro.ir at 16:46 +0330 on Sunday March 4th 2018 samples were kept in fiber glass tanks the fish was at a perpendicular angle to (2 tones capacity) for about two the ventral surface (at approximately months, under observation during 90º), until some blood entered into the acclimation in our research facility. The syringe or when the needle made average water quality conditions in the contact with the vertebral column (hard tanks during this period were impenetrable surface). Once the needle maintained at 26.5ºC, 5.7 ppm and 7.6 touched the vertebral column, it was for temperature, dissolved oxygen and withdrawn slightly, approximately 1 pH, respectively, and were confirmed mm, so that the blood vessel overlying optimal to maintain sound health of the the vertebral column could then be species. sampled easily and rapidly. This was Using an appropriate net, we done by gently pulling on the plunger randomly removed individual fish from by maintaining consistent pressure until the holding tank and immersed it into the desired quantity of blood is drawn anesthetic solution (containing 2.5 mL into the syringe. The collected blood 938 Argungu et al., A simple and rapid method for blood collection from walking catfish, Clarias …

sample was slowly but quickly and biochemical studies. All the fish in transferred into collection tubes this experimental trial (ranging in size (containing EDTA or heparin or treated between 75 g and 310 g) made 100% as may be desired per individual recovery and survived, thereafter to experimental set up). The sampled fish later be subjected to further was then transferred to a recovery tank, experimentation. containing clean, aerated water (also prepared before the commencement of Discussion the procedure), where it recovered fully According to MUAWC (2008), the and was swimming freely within 45-60 most popular technique for blood minutes. sampling from fish is the caudal vein The tube containing sampled blood method. Attempts were made by other was gently mixed with the anti- workers using this popular method, to coagulant (as the case may be) by sample blood from over ten individuals carefully turning it upside down to mix of C. batrachus in our laboratory, but the contents. The tubes were then kept were met with little success. These in a sampling box containing ice for attempts failed in spite of help offered further analysis. by experienced hands in blood collection from other species of catfish, Results using the caudal vein technique. This Using the presently proposed protocol, challenge may explain why some we successfully collected 0.5 mL of researchers previously working on the blood from each of the 24 fish (3 same species, C. batrachus, resorted to replicate fish per treatment, of 8 completely severing off the caudal treatments) during each of 6 hourly peduncle to obtain the desired quantity Downloaded from jifro.ir at 16:46 +0330 on Sunday March 4th 2018 time course sampling periods, within 24 of blood from the species (Kulshrestha hours (a total of 4 collections per fish, and Mandal, 1982; Maheswaran et al., each yielding a total of 2.5 mL of 2008; Kumar et al., 2010). Consistent blood). The fish sampled ranged in size with this speculation, some authors between 90 g and 160 g and were made reported sectioning the caudal peduncle up of equal numbers of male and (Srivastav and Srivastav, 1988), female individuals. Among these whereas others had to stun the animals sampled fish, over 75% (made up of (Raja and Sapkal, 2011) to collect some 20 individuals) recovered fully enough blood samples due to the and survived the blood collection. difficulty and stressful nature of the Similarly, we were also able to use this operation when conventionally same proposed procedure to collect 1 targeting the caudal vein in the species. mL of blood from each of 40 individual The procedure used in these trials fish (males and females) of Channa indicate that, not only is the popular striata species, during haematological method slow and stressful to C. Iranian Journal of Fisheries Sciences 16(3) 2017 939

batrachus, it is also cumbersome. That between respiratory and systemic those earlier workers neither mentioned circulations (Ishimatsu et al., 1979). A the survival rates after their procedure secondary circulation system has also nor used other conventional methods, is been identified in fish; an arterial- additional evidence to suspect that capillary-venous system derived from those methods were considered less the primary circulation which supplies convenient. The difficulty of blood the fins, tail and skin with blood. collection from C. batrachus could Capillaries are drained into a large therefore, be attributed to the small size collecting vein that flows towards the of caudal veins and peculiarities in the head. In salmonids, a “caudal heart” physiology of its cardio-vascular helps to force blood from the secondary system relative to the moderate size of capillaries system in the tail into the the species. caudal vein, driven by skeletal muscle The cardio-vascular system of the contractions. The venous blood is then walking catfish, C. batrachus is that of returned to the heart, by the posterior a primitive, air-breathing fish (Graham, cardinal vein, which is also located 1997), a similarity of this characteristic beneath the vertebral column. All the which it shares with snakehead fish, venous blood collects into one vessel Channa striata (Aliyu-Paiko et al., (Sinus venosus) before entering the 2010). These air-breathing species are heart. Therefore, skeletal muscle much unlike their typical water- contractions, vasodilators and capillary breathing counterparts in the circulation attraction play important roles in the systems. The system in water-breathing transportation of venous blood (Farrell fish is reported to be a single “in-series” et al., 2001; Sandblom and Axelsson, arrangement, in which blood from the 2007). Downloaded from jifro.ir at 16:46 +0330 on Sunday March 4th 2018 veins enters the heart via sinus venosus In “air-breathing” fish however, this with pressure below that in the clear separation is not in place, where atmosphere. The venous blood is then mixing of oxygenated and forced to the gills via the ventral aorta deoxygenated blood occurs in some by the heart. Upon oxygenation in the species. Among these, some species gills, the blood does not return to the have evolved breathing organs between heart like in mammals, but arterial the gills and systemic circulation so that blood is forced into the dorsal aorta that mixing does not occur, whereas in runs just beneath the vertebral column many others like Channa (and probably and to further be dispersed into the C. batrachus) the arrangement is such different tissues and organs via arteries, that oxygenated blood from the air- arterioles and the capillaries (Olsen, breathing organs is sent to the venous 2011). The heart pumps out the blood to system and returned to the heart, before the systemic circulation through the it is pumped to the systemic circulation respiratory organs, with clear separation (Satchell, 1976). Overall, these 940 Argungu et al., A simple and rapid method for blood collection from walking catfish, Clarias …

modifications due to the air-breathing the caudal vein difficult in the species. nature of the fish have affected the Added to this, vasodilators and skeletal arrangement of the central muscle contraction play important roles cardiovascular system of the species to on the pressure during transportation of show several unique deviations from venous blood (Sandblom and Axelsson, that of the typical teleost design 2007; Farrell et al., 2001) and proper (Ishimatsu, 2012). The most notable anesthesia (which is part of the blood among these deviations is the presence collection procedure) severely affects of anterior and posterior ventral aortae muscle contraction. This may be (Ishimatsu and Itazawa, 1983) as additional evidence to explain the lack opposed to the single dorsal aorta in of success in collecting blood from the water-breathing fish. Blood oxygen species using the popular protocol. pressure is the highest in the anterior Considering that C. batrachus is a cardinal vein, which receives threatened species (Hossain et al., oxygenated blood from the 2006; Ahmad et al., 2012) and is suprabranchial organ, and is lowered at reported to be vanishing in some parts the common cardinal vein and sinus of Asia (Binoy, 2010), further venosus when mixed with venous blood unnecessary waste of individuals cannot from posterior cardinal and hepatic be afforded, especially through veins, respectively (Ishimatsu et al., experimental designs which are 1979). According to Satchell (1976), developed with the sole purpose of the advantage of this drainage of maximizing blood collection. This oxygenated blood into the venous formed one of the important reasons for system is such that the lower pressure at the development of the present the peripheral end of the respiratory protocol, to make blood collection Downloaded from jifro.ir at 16:46 +0330 on Sunday March 4th 2018 capillaries, caused by their exposure to easier and more rapid. Our procedure the vis a fonte of the central veins rather was not only rapid and of minimal than the arterial pressure of the dorsal stress to the species, but is also non- aorta lowers the average pressure along lethal and does not require the help of their lengths. Therefore, the an assistant to restrain the fish or any combination of 1) presence of two sophisticated equipment such a “V” aortae, 2) the lack of anatomical board fish holder commonly used division in the venous system and 3) the during most of the existing protocols. absence of sino-artrial valves, which Considering the positive attributes according to Johansen (1971) play an mentioned above, the procedure important role in ventricular filling in described in our proposed protocol other teleosts, all contribute to lowering comply consistently with the ethical the pressure of blood in caudal veins of requirements of the Australian Code of “air-breathing” fish such as C. Practice for the Care and Use of batrachus, making blood collection at Animals for Scientific Purpose Iranian Journal of Fisheries Sciences 16(3) 2017 941

(MUAWC, 2008). The important enhancing blood sampling from the advantage which it has over other species for various investigations. conventional approaches being a one- operator procedure makes it suitable for Acknowledgment broader applications, where it is likely The authors gratefully acknowledge the to prove useful in collecting blood from contributions of the Department of different species. Lastly, the only Aquaculture, Faculty of Agriculture and limitation to the proposed protocol for Department of Biology, Faculty of now is that it was tested on only two Science, Universiti Putra Malaysia, species (Clarias batrachus and Channa Serdang, Selangor, Malaysia for striata). It is expected that operators providing materials and facilities for may wish to test the procedure on a this research. wide range of species, especially the primitive, air-breathing species, to References investigate its effectiveness and Ahmed, R., Pandey, R.B., Arif, S.H., applicability to the species targeted. Nabi, N., Jabeen M. and Hasnain, Blood collection from Clarias A., 2012. Polymorphic β and γ lens batrachus by an individual operator crystallins demonstrate latitudinal could be made simple and rapid when distribution of threatened walking carried out based on the procedure catfish, Clarias batrachus (Linn.) described in this protocol. This is a population in north-western. Indian fundamental achievement, considering Journal of Biological Science, 12(2), that the species is a promising 98-104. aquaculture candidate and one of the http://scialert.net/fulltext/?doi=jbs.20 economically important indigenous 12.98.104 Downloaded from jifro.ir at 16:46 +0330 on Sunday March 4th 2018 freshwater fishes in Southeast Asia. The Aliyu-Paiko, M., Hashim, R. and fact that the species is reported to be Shu-Chien, A.C., 2010. Influence of approaching extinction due to its dietary lipid/protein ratio on scarcity in the wild and competition survival, growth, body indices and with Clarias gariepinus, makes the digestive lipase activity in snakehead development of this protocol timely. (Channa striatus, Bloch, 1793) fry Therefore, any research practice or reared in re-circulating water system. cultural operation that duly contributes Aquaculture Nutrition, 16, 466-474. to further depletion of the species must Argungu, L.A., Christianus, A., be avoided, in favor of conservation Amin, S.M.N., Daud, S.K., Siraj, friendly efforts. This blood sampling S.S. and Aminur Rahman, M., methodology promises to support 2012. Asian catfish Clarias conservation efforts of this batrachus getting critically economically important fish by endangered. Asian Journal of Animal 942 Argungu et al., A simple and rapid method for blood collection from walking catfish, Clarias …

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