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Investigation of selected variables from the primary and secondary circulatory systems in rainbow trout… Ahlborn, Dorit 1992

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INVESTIGATION OF SELECTED VARIABLES FROM THE PRIMARY ANDSECONDARY CIRCULATORY SYSTEMS IN RAINBOW TROUT (Oncorhynchusmykiss).byDORIT AHLBORNB.Sc., The University of British Columbia, 1990A THESIS SUBMITTED IN PARTIAL FULFILLMENT OFTHE REQUIREMENTS FOR THE DEGREE OFMASTER OF SCIENCEinTHE FACULTY OF GRADUATE STUDIES(DEPARTMENT OF ANIMAL SCIENCE)We accept this thesis as conformingTHE UNIVERSITY OF BRITISH COLUMBIAJune 1992© Dorit Ahlborn, 1992In presenting this thesis in partial fulfilment of the requirements for an advanceddegree at the University of British Columbia, I agree that the Library shall make itfreely available for reference and study. I further agree that permission for extensivecopying of this thesis for scholarly purposes may be granted by the head of mydepartment or by his or her representatives. It is understood that copying orpublication of this thesis for financial gain shall not be allowed without my writtenpermission.(Signature)Department of  Animal &..if-t1C-02The University of British ColumbiaVancouver, CanadaDate ^9'7.2DE-6 (2/88)iiABSTRACTA series of experiments are reported which utilize acannulating method for the secondary circulation. Amodification of a new method for cannulating the secondarysystem in rainbow trout (Oncorhynchus mykiss) is described.A T-shaped catheter was placed inside the lateral cutaneousvessel, thereby allowing multiple samples to be taken fromthe same animal.Rainbow trout were cannulated in both the primary andsecondary circulatory systems. Repeated samples were takenover several days to determine the concentrations ofselected parameters. White blood cell numbers weresignificantly greater in the secondary system; however, theyalso varied much more than the cell numbers in the primarycirculation suggesting that blood cell composition changeswithin the secondary circulation. Concentrations ofselected parameters from the secondary circulation werecalculated as a percentage of the primary system: plasmacortisol was 60.2 ± 9.7%; total protein was 72.9 ± 4.9%,plasma glucose was 90.3 ± 11.4%, plasma chloride was 97.8 ±1.9%, and hematocrit was 0.5 ± 0.2%. Only glucose andchloride concentrations did not differ significantly betweenthe two systems. Adrenaline was determined to be 64.9 ±17.3% in the secondary compared to the primary circulation;however, since this hormone was detectable in only a fewsamples, this value may not be representative of the system.A sharp increase in the cortisol concentration of theprimary system 48 hours after operation was observed againapproximately two hours later in the secondary system,suggesting a slow mixing time between the primary andsecondary circulation.Fish were vaccinated with an intraperitoneal injectionof Vibrio anguillarum to investigate the possible role ofthe secondary system in immune function. As in the firstexperiment, the white blood cell numbers were significantlygreater and more variable in the secondary compared to theprimary circulation. The concentrations of selected plasmaconstituents in the secondary system were also determined aspercentages of the primary circulation: lysozyme was 114.9± 14.9%, cortisol was 51.4 ± 17.2%, total protein was 71.1 ±7.5%, glucose was 92.1 ± 11.8%, and chloride was 96.4 ±2.3%. Only total protein was significantly differentbetween the two circulatory systems. A low response in thehemolytic plaque assay and a negative outcome from antibodytiter tests in both circulatory systems, suggests that theadaptive (specific) immune response may not have beenstimulated.ivTABLE OF CONTENTS ABSTRACT^ iiTABLE OF CONTENTS^ ivLIST OF FIGURES viLIST OF TABLES^ viiiDEDICATION ixACKNOWLEDGEMENTS^GENERAL INTRODUCTION 1CHAPTER 1:A METHOD FOR CANNULATING THE SECONDARYCIRCULATORY SYSTEM IN RAINBOW TROUT^ 10INTRODUCTION^ 11MATERIALS AND METHODS^ 131) METHOD OF IWAMA AND ISHIMATSU^ 142) MODIFIED METHOD^ 18DISCUSSION^ 25CHAPTER 2:CHARACTERIZATION OF SELECTED VARIABLES FROMTHE PRIMARY AND SECONDARY CIRCULATION OFRAINBOW TROUT (Oncorhynchus mykiss)^ 28INTRODUCTION^ 29MATERIALS AND METHODS^ 32RESULTS^ 38DISCUSSION 50CHAPTER 3:INVESTIGATION OF SELECTED VARIABLES FROMTHE PRIMARY AND SECONDARY CIRCULATORYSYSTEMS IN SIX RAINBOW TROUT (Oncorhynchusmykiss) VACCINATED WITH Vibrio anguillarum^ 57INTRODUCTION^ 58MATERIALS AND METHODS^ 62RESULTS^ 68DISCUSSION 80CHAPTER 4:SUMMARY AND RECOMMENDATIONS^ 86REFERENCES^ 91APPENDICES 94viLIST OF FIGURESFigure 1.1) Iwama and Ishimatsus' method for inserting aT-shaped catheter into the lateral cutaneousvessel^ 15,16Figure 1.2) Modified method for inserting a T-shapedcatheter into the lateral cutaneousvessel^ 20,21Figure 2.1) Means ± s.e. of plasma cortisol (Ag/dL) fromthe primary and secondary circulation inrainbow trout^ 44Figure 2.2) Selected secondary variables expressed aspercent of primary circulation^ 45Figure 2.3) Means ± s.e. of total protein (g/dL) from theprimary and secondary circulation in rainbowtrout^ 47Figure 2.4) Means ± s.e. of plasma glucose (mg/mL) fromthe primary and secondary circulation in rainbowtrout^ 48Figure 2.5) Means ± s.e. of plasma chloride (mEq/L) fromthe primary and secondary circulation inrainbow trout^ 49Figure 3.1) Means ± s.e. of plasma lysozyme activity(Ag/mL) from the primary and secondary circulationin rainbow trout vaccinated two weeks earlier withformalin killed Vibrio anguillarum^ 74Figure 3.2) Selected secondary variables expressed aspercent of the primary circulation in rainbowtrout vaccinated two weeks earlier withformalin killed Vibrio anguillarum^ 75Figure 3.3) Means ± s.e. of plasma cortisol (Ag/dL) fromthe primary and secondary circulation inrainbow trout vaccinated two weeks earlierwith formalin killed Vibrio anguillarum^ 76Figure 3.4) Means ± s.e. of total protein (g/dL) from theprimary and secondary circulation in rainbowtrout vaccinated two weeks earlier withformalin killed Vibrio anguillarum^ 77Figure 3.5) Means ± s.e. of plasma glucose (mg/mL) from theprimary and secondary circulation in rainbowtrout vaccinated two weeks earlier withformalin killed Vibrio anguillarum^ 78vi iFigure 3.6) Means ± s.e. of plasma chloride (mEq/L) fromthe primary and secondary circulation inrainbow trout vaccinated two weeks earlier withformalin killed Vibrio anguillarum^ 79Figure A) Construction of a T-piece for cannulation ofthe lateral cutaneous vessel^ 100,101vi i iLIST OF TABLESTable 1.1) Summary of Iwama and Ishimatsu's method and themodified method for cannulating the lateralcutaneous vessel in rainbow trout^ 24Table 2.1) Hematological data from the primary andsecondary circulation of rainbow trout^ 41-43Table 2.2) Adrenaline concentrations and sample sizesfrom the primary and secondary circulation ofrainbow trout^ 46Table 3.1) Summary of hemolytic plaque assay and live whiteblood cell counts for six rainbow troutvaccinated two weeks earlier with Vibrioanguillarum^ 71Table 3.2) Hematological data from the primary andsecondary circulation in rainbow troutvaccinated two weeks earlier with Vibrioanguillarum^ 72,73To my father,You have inspired in me a desire for knowledge.ixxACKNOWLEDGEMENTS I would like to thank Dr. George K. Iwama for hissuggestions and support during the completion of this thesisand resulting degree. I greatly appreciate the time thatmy committee members spent in reviewing this manuscript.Thanks are extended to Dr. Alec G. Maule for hiscollaboration on chapters 2 and 3 which compare the primaryand secondary circulatory systems. My fellow graduatestudents and the research assistants from the Iwama labproved to be an excellent resource and they are warmlythanked. I am deeply indebted to my family and friends fortheir undying support, help and understanding during thecompletion of this degree, and especially to Boyd for hishelp and perseverance during even the most trying times.Thank you all.1INTRODUCTIONFish have a circulatory system that consists of both aprimary and secondary system (Vogel, 1985a). The primarysystem has been extensively researched while the secondarycirculation has not. Morphological descriptions of theteleost circulatory system referred to the secondary vesselsas either a lymphatic or venolymphatic system (Vogel andClaviez, 1981). However, tiny anastomoses connect thesecondary to the primary circulation throughout the body(Vogel, 1981); therefore using a strict definition (Shields,1972), it is not a lymphatic system. The secondarycirculation consists of an extensive vessel network that hasboth artery-like and vein-like structures (Mayer, 1917).The delicate vessels contain fluid at very low pressures(Vogel, 1985a); these combined features make the secondarysystem difficult to investigate. As a result, there hasbeen no published technique for obtaining multiple samplesfrom the same individual. In this thesis, a novel method isdescribed for cannulating the lateral cutaneous vessel fromthe secondary circulation and is subsequently used in twoexperiments.Several names have been given to the secondarycirculation resulting in a mixed assemblage of publishedmaterial. The term most used by the morphologists has beenlymphatics (Jourdain, 1868, 1880; Burne, 1926, 1929; Mayer,21917; Hans and Tubencka 1938; Kampmeier, 1969), because ofthe apparent absence of red blood cells, low pressures andthe resemblance of the vessel structures to mammalianlymphatics (Shields, 1972). Later, when red blood cellswere found within the vessels, there were several namesgiven to the system; the most common of which wasvenolymphatic (Cooke, 1980; Cooke and Campbell, 1980;Farrel, 1980; Olson and Kent, 1980; Rowing, 1981). Burne(1926, 1929) called the vessels 'fine' vessels, but Allen(1906) claimed that they were lymphatics. To add to thecontroversy, Wardle (1971) and Ellis and de Sousa (1974)believed that the red blood cells were an artifact ofsampling. The confusion over what to call this vesselsystem was well documented by Burne (1929) and untilrecently, lymphatic and venolymphatic were acceptedgenerally as being the appropriate names. A true lymphaticsystem, as in mammals, consists of extensive numbers oftubules that converge into a series of larger vessels andempty eventually into the primary system (Shields, 1972;Roitt, 1988). Vogel (1981) summarized that in severalteleost fishes, the ends of these tubules were actuallyconnected by minute anastomoses (10-14 Am in diameter) tothe primary system. The possibility of tiny connections wasfirst suggested by Burne (1929). He discovered that somecasts of the primary system had tiny projections whichcoincided with the ends of the extremely small vessels(tubules) that he called 'fine' vessels. Although he never3found a connection, he concluded that these vessels were infact an offshoot of the primary arterial circulation andthat only valves separated the two systems. Improvedmicroscopy provided conclusive evidence of these connections(Vogel, 1978, 1981) and showed that these interarterialshunts were located wherever there were primary arteries(Vogel, 1985a). Therefore, this vessel system is not a truelymphatic, but is in fact a secondary circulatory system(see Vogel and Claviez, 1981). This terminology will beadhered to throughout this thesis.It was also determined that the anastomoses havespecialized epithelial cells guarding the opening of thesecondary system. Microvilli protrude from these cells intothe lumen of the primary vessels (Vogel et al., 1976, Vogel,1981). These guard cells were found in Tilapia (Vogel etal., 1974) and rainbow trout (Vogel, 1978), but were notfound in the extensive non-respiratory vascular bed presentin the gills of smooth toadfish (Torquiginer glaber) (Cookeand Campbell, 1980). The microvilli are believed to beinvolved in the regulation of blood entry into the secondarysystem (Vogel, 1981). Their presence may aid in plasmaskimming (Krogh, 1959a) by acting as a filter and preventingentrance of red blood cells into the secondary system. Itis also possible that they are involved in sensory detectionof blood constituents, the presence or absence of componentscausing the anastomoses to open or contract.4Anastomoses are the same size as capillary vessels(Steffensen et al,. 1986). Capillaries have the ability tocontract and withstand enormous amounts of pressure (Krogh,1959b). It is possible that these anastomoses have similarcapillary-like characteristics. The ability to contractwould allow them to regulate fluid perfusion to certainareas (Vogel and Claviez, 1981). Anastomoses are oftenlocated close to nerve cells suggesting nervous control ofthe vessels (Vogel et al., 1973). Adrenaline, which isreleased during stress, prevents a decrease in efferentblood flow from the primary circulation in gills (Olson,1984), while Hughes et al. (1982) showed that adrenalineconstricted veno-venous anastomoses in the eel gill. On theother hand, acetylcholine, perfused through the gill beforea casting material, resulted in better filling of thesecondary (nutritive) vessels (Cooke and Campbell, 1980).If the anastomoses are closed during stress as suggested byVogel (1981, 1985a) and they are able to withstand highpressures, it would explain why researchers never foundconnections between the primary and secondary systems (seeBurne, 1929).Besides the anastomoses, there are two other areaswhere the secondary circulation directly connects to theprimary system. One is the caudal heart in the tail of fish(Vogel, 1985b) and the other area is located near the head(Burne, 1926; Kampmeier, 1969). These larger connections5are the termination points of the secondary system where thesecondary fluid drains into veins of the primary circulation(Kampmeier, 1969).It is believed that the extensive secondary circulationis a general feature of all fish (Jourdain, 1880; Vogel,1985a). The morphology of this circulation has been welldocumented (eg. Hewson, 1769; Allen 1906; Mayer, 1917;Burne, 1926, 1929) and is nicely summarized by Kampmeier(1969). Following is a short review of the location of thesecondary system in fish.Throughout the body, this system generally parallelsthe primary circulation (Burne, 1929; Vogel, 1981).Secondary vessels are found in the gills (see Vogel, 1978)and are connected to the central venous sinus (Nilsson,1986; Vogel, 1985a). Vessels supply the mouth mucousmembranes, skin (Burne, 1929) and peritoneum and are evenfound on top of the scales (Vogel, 1985a). There are alimited number of vessels located within the muscle tissue(Burne, 1929). No vessels are found in the cranial cavity(Allen, 1906; Vogel and Claviez, 1981). In the glasscatfish, only the secondary vessels are present in the fins(Steffensen et al., 1986). Wardle (1971) summarized themajor vessels as the longitudinal ventral, dorsal andlateral 'lymph' ducts. He concluded that in the plaice,these were just collecting sinuses that passed the fluid tothe neural lymph duct; in salmonids, however, this is not6the case since the neural duct is degenerate. The laterallymphatic trunk, now called the lateral cutaneous vessel, islocated directly beneath the skin, just above the lateralline (Allen, 1906; Kampmeier, 1969). Its accessibility hasmade it the vessel of choice for injections by manyresearchers (Allen, 1906). For a similar reason, thisvessel is used for cannulation (described in Chapter 1).The primary and secondary circulatory systems havesimilar vessel construction. Jourdain (1880) and Mayer(1917) found that there were afferent and efferentcomponents to the 'lymphatic' system. Fluid moved towardsthe periphery in arterial-like vessels (see: fine vesselsin Burne, 1926, 1929) and other vein-like vessels carriedthe fluid back, eventually converging into the larger vein-like collecting vessels. The arterial and venous componentsof the secondary system have thinner vessel walls than theprimary system, probably as a result of the lower pressure(Vogel, 1985a).The main pressure source for the primary and secondaryvessels is the heart (Vogel, 1985a, 1985b). The secondaryfluid is also moved by the respiratory pump in plaice(Wardle, 1971), and in other teleosts by body movements,muscle contraction and caudal hearts (Vogel, 1985b). Thevolume in the secondary system is about four times theprimary (Wardle, 1971). As a result, there is a much slowerfluid velocity and pressure in this system compared with the7primary circulation (Steffensen et al., 1986). Although thepressure in the secondary system is very low, there havebeen no measurements published that state the velocity(Vogel, 1985a).The secondary fluid is similar to blood. Fluidoriginates from the primary circulation and enters into thesecondary system via the anastomoses. An ultrafiltrate mayalso enter the secondary circulation from the extracellularspace in a similar manner as lymph formation in mammals(Shields, 1972; Roitt, 1988). Vogel (1985a) suggested thatblood constituents are present in similar quantities in thesecondary system. He explained further that the absence ofred blood cells is probably not normal and that in anunstressed fish the hematocrit should be greater in thesecondary, although still less than the primary circulation(Vogel, 1981). Fluid from both circulatory systems shouldbe similar in composition because the secondary directlycommunicates to the primary circulation at the anastomoses;however, the secondary fluid contains smaller quantities ofred blood cells and Vogel and Claviez (1981) called itplasma.Due to the absence of experimental techniques, there islittle information regarding the concentration of plasmaconstituents within the secondary system. This has resultedpartly from the system being difficult to investigate andpartly because, it does not appear to be as important as the8primary circulation. In the following thesis, a novelmethod is described for cannulating the lateral cutaneousvessel from the secondary circulation. Using this techniqueas well as a dorsal aorta cannula, characterization ofselected variables in both the primary and secondary systemswas accomplished in this work. Blood was repeatedly sampledthereby yielding selected primary and secondary circulatorymeasurements from the same individual over time.Due to the lack of experimental information, thefunction of the secondary circulation is not clear (Vogel,1985a). One of several possible roles of the secondarysystem may be an involvement in the immune system. To testthis, fish were vaccinated with Vibrio anguillarum. Twoweeks later, they were cannulated in both the primary andsecondary circulatory systems and the plasma analyzed forselected variables.The thesis is organized as follows: Chapter onedescribes the novel cannulating technique and its furthermodification for the lateral cutaneous vessel from thesecondary circulation in rainbow trout (Oncorhynchusmykiss). Chapter two presents the findings of an experimentdesigned to describe selected variables from the primary andsecondary circulatory systems in rainbow trout. To studythe possible role of the secondary circulation in the immunesystem, Chapter three presents experimental data from bothcirculatory systems of vaccinated rainbow trout. Overall9discussion, conclusions, and recommendations are presentedin chapter 4.CHAPTER 1:A METHOD FOR CANNULATING THE SECONDARY CIRCULATORY SYSTEM INTHE RAINBOW TROUT (Oncorhynchus mykiss)1011INTRODUCTIONThe use of indwelling catheters has greatly aidedresearchers in characterizing blood components from theprimary circulation in teleosts. The absence of acomparable long term cannulating technique for the secondarycirculation has prevented a similar identification ofsecondary plasma constituents. The aim of this chapter wasto describe a novel method for the cannulation of thelateral cutaneous vessel from the secondary circulation.Indwelling catheters are useful in providing long terminformation on blood constituents from the same animal. Acatheter enables the experimenter to sample blood from thesame fish without much disturbance, thereby providingsamples that are more representative of an unstressed fishthan if one was constantly handling the animal. Primarycirculation cannulas have been used since Smith and Bell(1964) developed a method to sample blood from free swimmingsalmonids. The technique has been improved and modified sothat now the dorsal aorta is routinely cannulated forobtaining blood samples from the primary system (Soivio etal., 1975).To date, the author is not aware of any publishedmethod describing a technique for inserting an indwellingcatheter into the secondary system. Previously Wardle(1971) sampled secondary plasma by inserting a syringe into12the neural duct of plaice. Ellis and de Sousa (1974)altered Wardle's technique by inserting a catheter into thisvessel in anesthetized fish; however, the cannula wasremoved after 4 to 5 mL of fluid had been collected. Thehandling required by these methods can severely stress thefish resulting in parameter values that may not be normal.The low pressure of this system is a major problem becauseinsertion of a catheter, similar to that used by Soivio etal. (1975) could theoretically block the flow. This problemwas overcome by Drs. G.K Iwama and A. Ishimatsu (personalcommunication) who designed a T-shaped cannula (seeappendix A) allowing normal, continuous flow through thevessel which is only interrupted during sampling. Thiscatheter is implanted into the lateral cutaneous vessel,whose proximity to the skin requires minimal surgery.Following is a report on Iwama and Ishimatsu's method ofinserting the T-piece as well as a modification developed bythe author.13MATERIALS AND METHODSFISHRainbow trout (Oncorhynchus mykiss) were obtained fromFraser Valley Trout Farms and allowed to acclimate for twoweeks at the University of British Columbia South Campusfacility and then another two weeks in the laboratory priorto experimentation. At both locations, the fish were heldin 1000 L tanks plumbed with Vancouver city tap waterdechlorinated with 5 ppm thiosulfate. Water temperatureranged from 8 to 9.5 °C. The fish were fed daily tosatiation using EWOS (Surrey, British Columbia) growerpellets, but were starved two days prior to surgery. Sex ofthe fish was not determined. A total of 25 animals werecannulated weighing 729.5 ± 18.4 (ranging from 604.0 to1026.0 grams).OPERATING PROCEDUREAll fish were anesthetized using tricainemethanosulfonate (100 ppm MS-222, Syndel) buffered with anequal part of sodium bicarbonate (NaHCO3, FischerScientific) in dechlorinated city tap water. The fish wereweighed and placed onto an operating table. The tableconsisted of a recirculating pump which irrigated the gillswith a maintenance dose of 50 ppm MS-222. A cooling coilregulated the water temperature, while an air stonemaintained an adequate oxygen concentration in the water.14The animal was placed on its right side (its head orientatedtowards the operators' left hand), exposing the left lateralline (see Figure 1.1a, 1.1b). A moist chamois was wrappedaround the animal to keep the skin wet, but leaving theoperation site free. In preparation for the operation, apolyethylene T-piece (see appendix A) was filled withheparinized Cortland saline lacking glucose (Wolfe, 1963).The operation then proceeded.1) CANNULATING METHOD OF IWAMA AND ISHIMATSU (personalcommunication).Approximately 4 cm from the head, a perpendicularincision (< 1 cm in length) was made across the lateral linecompletely severing the lateral cutaneous vessel in fish(Fig. 1.1c). The opening must be large enough to allow easyexposure of the vessel (Fig. 1.1d).^A prepared T-piece(Appendix^A) was cut so that the top of the 'T' wasapproximately 1.5 cm long. Heparinized saline was injectedinto the T-piece in excess so that drops were hanging fromthe ends (Fig. 1.1e). These drops ensured that no airbubbles were inside the catheter; any air that enters intothe secondary system can cause blockage of the vessel. Theskin anterior to the cut was lifted using forceps and the T-piece was inserted into the exposed vessel (Fig. 1.1f).exposure of the vessel (Fig. 1.1d).^A prepared T-piece(Appendix^A) was cut so that the top of the 'T' wasapproximately 1.5 cm long. More saline was injected into15Figure 1.1, following page) The method used by Iwama andIshimatsu to implant a T-shaped catheter (T) into thelateral cutaneous vessel (LCV): a) view of the fish as it isoriented on the operating table, b) enlarged view of theoperating site, approximate position of LCV, LL = lateralline, c) an incision (I) through the LCV, other scales aredeleted for visibility, d) view into the incision, showingthe completely severed vessel, e) T filled with heparinizedsaline, D = drops of saline, f) insertion of T into the LCV,g) uninserted part of catheter bent using tweezers (notshown), h) catheter inside the vessel, i) stitches closingthe incision (S1) and sealing the catheter-vessel complex(S2), an anchoring suture is located 3 cm above the LL (notshown).1617the catheter until a drop hung off of the uninserted end(Fig. 1.1f). Using forceps, this side was then bent (Fig.1.1g), the skin on the posterior side of the incision waslifted and the catheter released into the vessel (Fig.1.1h). This release must be gentle and fairly slow so as tominimize vessel damage.Sutures (silk from Ethicon, Inc.) were tied around thevessel and catheter on both sides of the cut (Fig. 1.1i).The stitches must be made deep enough so that they areunderneath and do not puncture the vessel, yet theunderlying muscle should not be penetrated too much. Theknots also need to be tied correctly. If they are tooloose, they will not seal the vessel-catheter complex, tootight and they can decrease the catheters' lumen. Thesutures serve two functions: 1) they secure the catheter inplace and (2) reduce the possibility of leakage. Saline wasinjected into the catheter to test for leakage from thesevered vessel. If there was leakage, another suture wasplaced further from the incision towards the end of the T-piece. Two stitches were used to close the incision (Fig.1.1i) and a final suture (approximately 3 cm above thelateral line) anchored the catheter in place. Blockage ofthe catheter was tested by placing the cannula below thefish and allowing the fluid to drain from the secondarysystem.Any bubbles visible inside the catheter were removed at18this time. The author was able to force bubbles out of thesystem by moving them along the cannula. Most of thecatheter was kept below the fish except for a small sectionthat was closest to the bubbles. The tubing was gentlytapped causing the bubbles to loosen and rise to the highestpoint, moving them away from the 'T' part of the catheter.This was continued until all bubbles were removed from thecannula. A syringe with heparinized saline was thenattached and the fluid flushed back into the system therebycleaning out the catheter. The end of the catheter wassealed by melting and then pinching the plastic together.The fish were placed in black Plexiglass gangboxes andallowed to recover from the operation. These boxes weresupplied with an adequate flow of dechlorinated city tapwater.2) MODIFIED LATERAL CUTANEOUS VESSEL CANNULATION.The author found that the procedure of Iwama andIshimatsu (used in Chapter 2) caused severe trauma at theoperating site. Of concern was the size of the incision,the excessive pulling on the skin required to insert thecannula, the number of stitches, and the act of putting thestitches in after having inserted the catheter. Bending thecatheter and then releasing it into the vessel was anotherpotential source of problems. The secondary vessel walls19are very thin (Vogel, 1985a) and if the catheter angle isnot perfect, it can damage, or even puncture the delicatewall thereby ending up in the surrounding tissue and notwithin the vessel.As a result of these concerns, the author modifiedIwama and Ishimatsu's method in the following way (used inChapter 3). Instead of one large incision, two cuts weremade 3/4 to 1 cm apart, just above the lateral line(Fig.1.2c). These cuts were only about three millimeterslong and ran perpendicularly to the lateral cutaneousvessel. The incisions penetrated only 1/2 to 3/4 of thevessel, leaving the side of the vessel farthest from theskin intact (Fig. 1.2d). A suture was started at each cutby inserting a needle through the skin on one side of theincision, running the needle underneath the vessel and thenthrough the skin on the other side of the cut. No knotswere tied, instead, the silk thread (about 6 cm in length )was left lying on the side of the fish (Fig. 1.2e). Oncethe catheter was in place, the knots could be tied withoutdifficulty.A catheter was measured and cut so that the top of the'T' was at least 50% longer than the distance between thetwo incisions (2/3 of this length must be on the side of the'T' inserted in the anterior cut, see Fig. 1.2f). Thisensured that approximately 25% of the catheter would be inthe vessel on both sides. A cannula that was too small in20Figure 1.2, next page) The modified method for implanting aT-shaped catheter into the lateral cutaneous vessel. SeeFigure 1.1 for abbreviations: a) view of the fish as it isoriented on the operating table, b) enlarged view of theoperating site, approximate position of LCV, c) 2 smallincisions into the LCV about 3/4 cm apart, d) view of theLCV, note that the vessel is not completely severed, e) Silkthread (S) for sutures, threaded underneath the LCV, f) Tfilled with heparinized saline, note that the top of the Tis of unequal length, g) insertion of T into the LCV at theanterior cut, h) T fully inserted into the LCV, I) suturestied thereby sealing the catheter-vessel complex, the endsof the sutures are fastened around the catheter bodyanchoring the cannulae in place. Another suture (3 cm abovethe LL) is tied to secure the catheter (not shown).Figure 1.2)®2122relation to this distance could slip out of the vessel ifthe fish was able to bend its body.As in the former method, the skin from the left side ofthe anterior cut was gently lifted using tweezers. The T-piece, with a drop of saline hanging from it (Fig. 1.2f),was inserted into the vessel keeping it as parallel aspossible to the lateral line and especially to the bodysurface. The catheter was slid all the way in until the 'T'structure stopped it (Fig. 1.2g). Saline was again injectedinto the catheter until a drop was hanging from theuninserted side (Fig. 1.2g). The skin on the right-hand-side of the posterior incision was lifted in the same mannerand the cannula gently slid into the opening until an equalamount of tubing was under the skin at both incisions (Fig.1.2h). This is one of the major differences between the twomethods. The modified method requires no bending of thecatheter or excess stretching of the incision site to allowthe second insertion of the catheter.Once the catheter was in place, the sutures weresecurely fastened around the cannula, thereby closing theincisions and preventing leakage from the cuts. The ends ofthe sutures were also tied around the vertical part of the Tto anchor the T-piece in place (Fig. 1.2i). The dualfunction of these stitches (sealing the incision andanchoring the catheter) resulted in a decrease in the numberof sutures required as compared with the first cannulating23method.From this point, the modified cannulating procedure wasthe same as the former method. Saline was injected into thecatheter to examine for leakage. Any leakage was againeliminated by tying a suture completely around the catheterand vessel. If no visible leaks were present, fluid wasallowed to drain out of the vessel and any air bubbles wereremoved. By infusing saline into the system, the catheterwas flushed clean. To check that the catheter was in thevessel, a bolus of saline was observed as it moved under thelateral line; the skin lifting slightly as the salinetraveled through the vessel. A final suture was placedabout 3 cm dorsally of the lateral line anchoring thecatheter in an upside-down T position. After completion ofthe operation, the fish were placed inside black Plexiglassgangboxes to recover from the surgery.24IWAMA AND^MODIFIEDISHIMATSU# INCISIONS^ 1^2 (approx. 3/4 cmapart)INCISION SIZE^< 1 cm^0.3 cmINCISION DEPTH^severing the^1/2 through vesselvesselSKIN STRETCHING^yes minimalCATHETER BENDING yes^ noINSERTION OF^after cannula^before cannulaSUTURES insertion insertion# OF SUTURES 4^ 2ANCHORING SUTURE^yes (1 suture)^yes (1 suture)LENGTH OF^36.2 ± 1.4 min.^36.7 ± 1.7 min.OPERATIONTable 1.1) Summary of Iwama and Ishimatsu's method and themodified method for cannulating the lateral cutaneous vesselin rainbow trout (Oncorhynchus mykiss).25DISCUSSIONAs a result of technical problems, the secondarycirculation has not been researched as extensively as theprimary system. No techniques have previously beendeveloped for the implantation of an indwelling catheter inthe secondary system. A method and modification forcannulating the secondary lateral cutaneous vessel weredescribed in this chapter (Table 1.1). The cannulatingprocedure of Iwama and Ishimatsu used only one incision tointroduce a T-shaped catheter. To overcome several of theproblems that were encountered while using this method, theauthor modified their technique. Two incisions, of smallerlength and decreased depth reduced the amount of trauma atthe operating site. These incisions reduced the lifting ofthe skin that was required to insert the cannula, andcatheter bending was completely eliminated. Preparing thesutures before the catheter was in place also eliminated thepossibility of sewing too deeply into the underlying muscle,or piercing the vessel since the cannula did not obstructthe view. The absence of the cannula also prevented vesseldamage resulting from the catheter moving while the needlewas inserted through the tough skin. The average length oftime to perform the operations (when both the dorsal aortaand lateral cutaneous vessel were cannulated) were similarfor both methods, they were 36.2 ± 1.4 and 36.7 ± 1.7minutes (mean ± 1 s.e.) for Iwama and Ishimatsu's method and26the modified method, respectively.Although the modified method decreases some of theproblems that were encountered, the procedure needs furtherdevelopment.^Clotting was the major problem experiencedduring cannulation and subsequent sampling.^This arosepartly because of an inflammatory response caused by injury(the incision) and the presence of a foreign object (thecatheter) (Playfair, 1984). Since this is a low pressuresystem clotting occurs more readily than in a high pressuresystem (Krogh, 1959a).Following are several suggestions which may helpalleviate the occurrence of clotting within the lateralcutaneous vessel after operation. Once the T-pieces aremade, they should be stored in heparinized saline so thatthe polyethylene tubing will absorb some of the heparin. Intheory, heparin will leach out of the implanted catheterthereby reducing the amount of clotting occurring on oraround the cannula. After the top of the T-piece has beenmeasured and cut to the correct length, the ends of the T-piece should be quickly run through a flame. The slightmelting of the tubing will result in a smoother surface.This may decrease the frequency of cells rupturing as theycome into contact with the catheter. It is also possiblethat vessel damage resulting from insertion of the cathetermay be reduced.27The problem of sealing the vessel system once thecatheter is in place may be more readily solved using atissue glue. This would have the added benefit of securingthe catheter and decreasing the number of stitches;therefore, trauma at the operation site may be reduced.Further work is required to produce a cannulating techniquefor the lateral cutaneous vessel that is as easily employedas a dorsal aorta cannulation.CHAPTER 2:CHARACTERIZATION OF SELECTED VARIABLES FROM THE PRIMARY ANDSECONDARY CIRCULATION OF RAINBOW TROUT (Oncorhynchusmykiss).2829INTRODUCTIONThe secondary system, considered at one time to be alymphatic system, may have been overlooked, or deemedunimportant while investigating the primary circulation.Interested researchers may have been discouraged by thesecondary system's morphology; the low pressure and delicatevessels collapsed when cut (Kampmeier, 1969). For thesereasons, published material concerning secondary fluidcomposition is scarce.The extensive secondary vessel network is connected byanastomoses to the primary circulation. Vogel (1985a)stated that these connections allow for similarities in theblood constituents of both systems. Wardle (1971) found thesecondary fluid to be clear with a composition mimicking theblood and plasma of the primary circulation. As a result ofsimilar observations, Vogel and Claviez (1981) proposed thatthis fluid be called plasma. However, the ability of theanastomoses to contract and decrease their lumen size,results in the secondary system being disconnected from theprimary circulation and may cause changes in theconcentration of blood variables.Red blood cells are found in very small concentrationswithin the secondary system, resulting in measuredhematocrit values that are 1-2% packed cell volume (Vogel,1985a). Wardle (1971) and Ellis and de Sousa (1974) stated30that the presence of red blood cells within the secondarycirculation was caused by contamination from the samplingsite. However, other researchers (see general introductionfor details) found the system to contain large quantities ofred blood cells that were not artifacts from sampling.Vogel (1985a) expected that in an unstressed fish, the redblood cell content within the secondary circulation shouldbe greater than usually observed, but still less than theprimary system. Low hemoglobin and red blood cell contentsuggest that the secondary system is not important foroxygen transport, but may provide nutrients to the skin(Steffensen et al., 1986).Besides a limited number of red blood cells, there arealso other cells present within the secondary circulation.Ellis and de Sousa (1974) determined that in a single samplefrom the neural duct tube in anesthetized plaice(Pleuronectes platessa), blood cell types were the same, butin a lower concentration than in the primary circulation(lymphocytes: 8X10 3 ; neutrophils 1X10 3 ; thrombocytes 12X10 3 ;and occasional monocytes (units were not mentioned in thispaper, but are most likely cells/mL)). On the other hand,Wardle (1971), sampling from the same location, found normallevels of mobile leucocytes and thrombocytes. There is somediscrepancy in the actual cell numbers present in thesecondary circulation.In his investigation, Wardle (1971) also analyzed other31variables. He determined that protein from the neural ducttube in plaice was 80.4% of blood plasma, and had noelectrophoretic differences. There seemed to be the samelactate levels present in the secondary as in the primaryplasma. Both he and Burne (1929) found blood clots ofsimilar appearance in the primary and secondary circulatorysystems.When the lateral cutaneous vessel cannulation(described in Chapter 1) is combined with a primary systemcannulation, it is possible to perform comparative studiesbetween the primary and secondary circulatory systems. Theobjective of the following experiment was to determinebaseline values for variables that are routinely sampledfrom the primary circulation. These variables werehematocrit and differential cell counts and plasma cortisol,adrenaline, total protein, glucose and chlorideconcentrations from both systems over time in consciousrainbow trout (Oncorhynchus mykiss).32MATERIALS AND METHODSFISHRainbow trout were obtained from the same trout farmand maintained in the same manner as those in Chapter 1:Materials and Methods. The experiment took place in Juneand July of 1991. The fish were fasted at least two daysprior to surgery. Fifteen animals, weighing 698.9 ± 11.8 g(mean ± 1 s.e.) , were cannulated in the primary andsecondary vessels.CANNULATIONSa) Primary circulation:The dorsal aorta was cannulated using a polyethylenecatheter (PE 60, Clay Adams Intermedic) following theprocedure of Soivio et al. (1975). The aortic cannulationalways preceded the secondary cannulation.b) Secondary circulation:The lateral cutaneous vessel was cannulated accordingto the method of Iwama and Ishimatsu, (see Chapter 1:Materials and Methods, part 1 (Method of Iwama andIshimatsu)).EXPERIMENTAL PROCEDURE AND SAMPLINGThe fish were allowed to recover in black plexiglassgangboxes for 24 hours following the operation and weresampled according to the following schedule: sample A was33obtained 24 hours after surgery; sample B was obtained 48hours after surgery; sample C was obtained 50 hours aftersurgery; sample D was obtained 52 hours after surgery;sample E was obtained 54 hours after surgery; sample F wasobtained approximately 72 hours after surgery; sample G wasobtained approximately 96 hours after surgery. Samplingoccurred while the animals were in the gangboxes and thecatheters had been threaded through a hole in the lid of thegangbox. Sampling continued from each fish until fluid fromeither catheters stopped flowing, or the animal died. Onefish was sampled during the operation; however, it died soonthereafter. For this reason, other fish were not sampledduring surgery.Both the primary and secondary circulatory samples weresimultaneously obtained by gravity into 1.5 mL Eppindorftubes. An initial 30 to 40 AL was dripped to waste toremove saline present from the catheter. After sampling wascompleted, an equal volume of heparinized saline was slowlyflushed back into the system replacing the fluid (Nichols,1987) and cleaning the catheter. Samples were spun down for4 minutes in a Micro Centrifuge (Johns Scientific Inc.,Toronto, Ontario, Canada). Plasma was removed, placed intoEppindorf tubes and immediately stored at -80 °C untilanalyzed for the variables listed below. At each samplingtime, hematocrit was measured and a blood smear was made fordifferential cell counts.34ANALYSESHEMATOLOGYHematocrit was measured by collecting blood into aheparinized capillary tube and reading the packed cellvolume (Langan and McIntyre, 1981; Randall, 1970). Thecapillary tubes were spun at 11,500 revolutions per minutefor 4 minutes in a micro hematocrit centrifuge(International Equipment Co., Needham Heights, Ma., U.S.A).Blood smears (one slide per circulatory system persampling period) were stained using Diff Quick (BaxterDiagnostics Corp., Canlab Division, Mississauga, Ont.,Canada) which gives results similar to a Wright-Giemsastain. Cells were counted using a 1000X oil immersion lenson a light microscope (Jena Med-2, Zeiss). Red blood cells,lymphocytes, neutrophils and thrombocytes were counted usingYasutake and Wales (1983) and Lehmann (1988) as references.For the primary circulation, nine fields were counted(average red blood cells was 960/slide ranging from 418 to1464). For the secondary circulation, however, the cellcounts were not as easy. Some of the slides had very fewcell numbers, so the whole slide was counted. Other slideshad greater numbers of cells that were spread out. Thevarying number of cells present on the slides made itimpossible to standardize the counts by reading a specifiednumber of fields. As a result, a range of 4 to 725 cells(of all types) were counted per slide. For both systems,cell numbers were converted to percentages of total counted35cells.PLASMA CORTISOLA radioimmunoassay (Coat-a-count: Cortisol, Assay #259,Baxter Diagnostics Corp) which uses competitive bindingbetween the plasma cortisol and 1251 tagged cortisolmolecules was used to determine plasma cortisolconcentration. This kit has a detection level of 0.2 gg/dLand has been used for investigations of cortisolconcentrations in salmonids (Redding et al., 1984).PLASMA ADRENALINEAdrenaline was analyzed by high pressure liquidchromatography (HPLC) following the procedure of Woodward(1982). The adrenaline molecules were extracted from theplasma by combining 200 AL plasma, 10 mg aluminum oxide(BAS, West Lafayette, Indiana), 400 AL of 2 M tris/EDTA and40 AL 3,4 dihydroxy benzylamine hydrobromide (DHBA) (seeAppendix B for solutions). As a result of the large samplesize required for adrenaline analysis, only one extractionwas performed on the samples. Following several washes witha 0.2% solution of Tris/EDTA (a 10:1 dilution of 2 Mtris/EDTA adjusted to pH 8.6), the adrenaline molecules wereresuspended in 125 AL of a solution containing 100 gL ofglacial acetic acid (BDH), 50 AL of 10% sodium disulfite(BDH) and 50 AL of 5% EDTA (BDH) diluted to 10 mL withdeionized water. All reagents were HPLC grade. The36resuspended adrenaline was then stored at -80 °C untilanalyzed by HPLC (Shimadzu Scientific Instruments Inc.,Maryland, U.S.A.; using a Waters Plasma Catecholaminecolumn, Division of Millipore, Milford, MA, U.S.A.).TOTAL PLASMA PROTEINThe Lowry method was used to determine the totalprotein concentration of plasma (Alexander and Ingram, 1980;Kit #5656, Sigma). Samples were analyzed in duplicate andread on a U.V. spectrophotometer (Shimadzu) at 750 nm.PLASMA GLUCOSEUsing ortho-toluidine (Sigma) in a modified glucoseassay (Wedemeyer et al., 1990), the samples were measured induplicate. Ten pL of plasma was combined with 3.5 mL ofortho-toluidine and heated for 10 minutes in a boiling waterbath. The reaction occurs when glucose condenses witharomatic amines to form colored glycosylamines (Burrin andAlberti, 1990). The developed color, which is stable for 30minutes, was read on a U.V. spectrophotometer (Shimadzu) ata wavelength of 635 nm.PLASMA CHLORIDEChloride analysis (done in duplicate) was performedusing a chloridometer (Haake Buchler Instruments, Inc.)which measures the reaction between silver and chlorideions.37STATISTICAL TREATMENTResults from the primary and the secondary circulatorysystems were subjected to analysis of variance followed by aTukey test (Sokal and Rohlf, 1981) to determine where, ifany, there was a significant difference between means withineach group. For each variable, the overall mean from eachcirculatory system was determined and a paired t-test wasperformed to compare the overall means between the twocirculatory systems. For selected variables, the overallmeans were also used to determine the secondaryconcentrations as a percentage of the primary circulation.Further analysis used paired t-tests to compare the groupaverages at individual sample periods. A computer aidedstatistics program (SYSTAT, Wilkinson, 1988) was used toperform the analysis, while Larkin (1975) and Runyon (1985)were used as references for statistical methods.38RESULTS During the experiment, the average sampling time was8.4 ± 1.7 and 35.2 ± 6.0 minutes (mean ± 1 s.e.) for theprimary and secondary circulations respectively. Clottingin the secondary circulation resulted in a fast reductionof sample sizes. Sampling from the secondary system wasmade very difficult by the presence of two main types ofclots. One type had the appearance of "fluffy" white ballsthat when magnified, appeared to be cell debris. The othertype of clot was almost invisible; one only noticed thatthese were present when no fluid dripped from the catheter.They were long elastic cable-like formations that oftenfilled the length of the catheter. Unfortunately these werenever examined under the microscope so their exact naturewas not determined. This debris could often be removed withcareful diligence; however, it required opening the gangboxand disturbing the fish.The decreasing trend in the primary systems' hematocritwas also observed in the hematocrit of the secondarycirculation (Table 2.1). The difference between the twosystems was highly significant at all sample periods. Thehematocrit of the secondary system was 0.5 ± 0.2% of theprimary circulation. The secondary system showed a largefluctuation in the number of red and white blood cell types(Table 2.1). This differed greatly from the primarycirculation where the quantities of the various cell types39did not change greatly. Although the main types ofleucocytes counted were lymphocytes, neutrophils andthrombocytes, there was also another unidentified form foundmainly within the secondary circulation. These cellsconsisted of a dark (almost black) nucleus, with no visiblecytoplasm (using a Wright-Giemsa stain). Since there werelarge quantities of these cells present, they were countedand called 'unknown' cells (see Table 2.1).The cortisol concentration in the primary system wassignificantly higher than the secondary (Fig 2.1). After aninitial increase in cortisol occurring at time B (48 hoursafter surgery), the cortisol concentration decreased in theprimary circulation. A more gradual increase was seen inthe secondary cortisol concentration which eventuallyreached the same level as the primary system at time C(approximately 2 hours after B). The cortisol concentrationin the secondary was 60.2 ± 9.7% of the primary circulation(Fig. 2.2).Not all the samples could be analyzed for adrenalinesince this assay required 200 AL of plasma (Table 2.2).Unfortunately many of the extracted samples were belowdetection levels for adrenaline. Averaging the values,however, resulted in the adrenaline concentration in thesecondary circulation being 64.9 ± 17.3% of the primarysystem and the difference was not statistically different(Fig. 2.2).40Total plasma protein concentration in the primarycirculation was significantly higher than in the secondarysystem (Fig. 2.3). Further analysis revealed that thedifferences were at times A (day 1 after surgery) and B (day2 after surgery). Total protein concentration from thesecondary circulation was 72.9 ± 4.9% of the primary system(Fig. 2.2).Glucose concentration was similar in both circulatorysystems; there was no statistical difference between the twogroups (Fig. 2.4). The glucose concentration of thesecondary circulation was 90.3 ± 11.4% of the primarycirculation (Fig. 2.2).Chloride concentrations and trends were similar in bothsystems (Fig. 2.5). The primary concentration was slightlygreater than the secondary, but the differences were notsignificant. The chloride concentration of the secondarysystem was 97.8 ± 1.9% of the primary circulation (Fig.2.2).41Table 2.1) Cell counts and hematocrit (mean ± 1 s.e.) fromthe primary and secondary circulation in rainbow trout.Statistical analysis was only performed on the raw data.There were no significant differences within each group. Asignificant difference was found between the overall meansof lymphocytes (P<0.01); thrombocytes, unknown cells,neutrophils, red blood cells, and hematocrit (P<0.001).Paired t-tests between the two circulatory systems alsorevealed differences at the individual sampling periods: a= (P<0.001), " = (P<0.01), c = (P<0.05), z = insufficientdata.neutro = neutrophil; thromb = thrombocyte; lympho =lymphocyte; unknown = cells that could not be categorized,but were probably thrombocytes and/or small distortedlymphocytes; WBC = white blood cells; RBC = red blood cells;HCT = hematocrit.Time A = 24 hours after surgery; B = 48 hours after surgery;C = 50 hours after surgery; D = 52 hours after surgery; E =54 hours after surgery; F = 72 hours after surgery; G = 96hours after surgery.42Table 2.1)AVERAGE CELL COUNTSSample^Size^Neutro Thromb Lympho^Un-^Total^Total(n) known WBC^RBCPRIMARY CIRCULATIONA^14^1.3^0.4b^4.0^1.0b^1.7^936.1a^0. 0.2 0.7 0.9 0.4 62.2B^11^0.9^0.7^2.7^0.0z^1.1^871.0a^0.5 0.3 0.5 0.0 0.2 86.2C^6^0.4^0.0^1.9^1.5z^1.0^901.0b^0.3 0.0 0.8 1.3 0.4 57.9D 5^0.6^0.3c^2.2c^0.3c^0.9^893.8a^0.4 0.2 0.5 0.3 0.2 82.5E 4^0.9^0.0z^3.3^2.5^1.7^938.3"^0. 0.0 1.2 2.5 0.7 68.0F^4^1.3^0.0z^3.3^2.8^1.8^1051.0c^0.9 0.0 1.8 2.8 0.8 146.2G 1^0.0z^0.0z^3.0z^0.0z^0.8z^1031.0z0.8SECONDARY CIRCULATIONA^9^14.4^18.3^5.3^64.3^25.6^66.2^6.3 5.1 0.9 13.7 5.4 23.7B 9^24.3^5.3^5.2^33.2^17.0^97.1^10.7 2.2 1.8 9.7 4.1 54.4C^6^8.6^9.4^2.4^33.0^13.4^256.8^3.7 2.3 0.5 16.0 4.6 130.6D 5^35.3^13.5^15.5^27.7^23.0^174.0^14.0 4.3 4.5 8.7 4.5 93.6E 5^74.8^13.0^20.4^31.6^35.0^242.6^41. 3.8 13.0 14.3 11.9 134.2F^2^20.0^2.0^17.5^15.0^13.6^407.5^4.0 2.0 4.5 13.0 3.8 107.5G 0Table 2.1 continued)AVERAGE CELL COUNTS AS PERCENTAGE OF TOTAL CELLSSample^Size (n)^Neutro^Thromb LymphoUn-knownRBC HCTPRIMARY CIRCULATIONA^14 0.1 0.04 0.4 0.1 99.3 31.110.03 0.02 0.7 0.1 0.2 2.4B^11 0.13 0.09 0.3 0.0 99.5 26.7a0.06 0.04 0.05 0.0 0.1 2.5C^6 0.04 0.0 0.2 0.14 99.6 29.0a0.03 0.0 0.09 0.13 0.1 3.1D^5 0.06 0.03 0.3 0.03 99.6 27.000.04 0.02 0.07 0.03 0.1 3.8E^4 0.1 0.0 0.4 0.3 99.2 25.000.06 0.0 0.14 0.3 0.5 3.7F^4 0.1 0.0 0.3 0.2 99.4 22.710.08 0.0 0.1 0.2 0.3 4.3G^1 0.0 0.0 0.3 0.0 99.7 28.01.._SECONDARY CIRCULATIONA^9 13.4 11.8 4.3 40.6 29.8 0.08.8 3.3 1.2 7.9 7.9 0.0B^9 19.5 9.1 8.7 27.8 34.9 0.76.9 4.3 4.6 7.6 10.8 0.4C^6 5.2 8.7 6.1 17.6 62.4 0.73.9 4.3 4.8 7.2 12.9 0.5D^5 22.9 9.5 6.9 19.3 41.5 0.611.3 5.1 1.4 8.0 15.7 0.4E^5 28.0 4.4 7.0 12.1 48.5 0.415.2 1.3 4.1 5.6 20.3 0.2F^2 4.4 0.3 3.8 2.7 88.7 0.00.4 0.3 0.1 2.1 1.9 0.0G^04325M Primary circulationF-7 Secondary circulation205A^B^C^D^E^F^GSAMPLING PERIODSFigure 2.1) Means ± 1 s.e. of plasma cortisol (Ag/dL) fromthe primary and secondary circulation in rainbow trout.Numbers indicate sample sizes. The overall means from thetwo groups are significantly different from each other(P<0.05). Sample times are the same as described in Table2.1.4411010090807060504030201045Cortisol Glucose^total^Chloride AdrenalineproteinSELECTED SECONDARY PARAMETERSFigure 2.2) Selected secondary variables expressed aspercent of the primary circulation. Statistical differencesbetween the two circulatory systems are indicated by **(P<0.001) and * (P<0.05).46SAMPLE A B C D E F GPRIMARY CIRCULATIONSIZE (n) 6 6 1 3 1 1 1ADREN. 2.689 6.420 3.09 3.997 13.374 1.98 2.7919(nM) 0.891 3.054 0.934 -SECONDARY CIRCULATIONSIZE (n) 2 3 1 1 0 1 0ADREN. 2.531 2.801 7.16 1.51 2.12(nM) 0.987 1.477 -Table 2.2) Means ± 1 s.e. of adrenaline (nM) from theprimary and secondary circulation in rainbow trout.Statistical analysis between the overall means revealed nosignificant difference. Sample times are the same asdescribed in Table 2.1.15 12Primary circulationV/I Secondary circulation15Prk A471210A^B^C^D^E^F^GSAMPLING PERIODSFigure 2.3) Means ± 1 s.e. of total protein (g/dL) from theprimary and secondary circulation in rainbow trout. Numbersindicate sample sizes. There was a significant difference(P<0.001) between the two systems. * indicates asignificant difference between the groups (P<0.05). Sampletimes are the same as described in Table 2.1.400 Primary circulationSecondary circulation48350\VA300iiEo' 250Ei.,..1U)0 200UD___JCD< 150CT)<__IQ_10050A^B^C^D^ESAMPLING PERIODSF^GFigure 2.4) Means ± 1 s.e. of plasma glucose (mg/mL) fromthe primary and secondary circulation in rainbow trout.Numbers indicate sample sizes. Statistical analysisrevealed no significant differences between or within thetwo systems. Sample times are the same as described inTable 2.1.49140Primary circulationcirculationSecondary130120 152612 6014 12U^110 3(f) 40_10090 .14 AA^C^D^E^FSAMPLING PERIODSFigure 2.5) Means ± 1 s.e. of plasma chloride (mEq/L) fromthe primary and secondary circulation in rainbow trout.Numbers on graph indicate sample sizes. Statisticalanalysis revealed no significant difference between orwithin the two systems. Sample times are the same asdescribed in Table 2.1.50DISCUSSIONLong sampling times are necessary to collect samplesfrom the secondary system. Ellis and de Sousa (1974), usinga peristaltic pump, required two hours to collect 4-5 mLfrom the neural duct tube of anesthetized plaice. It ispossible that constituents such as cortisol and adrenaline,within the resulting samples, may break down as they are notprocessed as quickly as samples from the primarycirculation. This could explain the overall lowerconcentrations of some of the plasma constituents within thesecondary circulation (see Fig. 2.2). Wardle (1971) foundprotein concentration to be 84% in the "lymph" of plaice.Although he did not mention the length of sampling time, itis assumed that his procedure (inserting a syringe into theneural duct tube and applying a negative pressure) wasquicker than sampling by gravity as in this experiment.Another explanation for lower constituentconcentrations within the secondary circulation is relatedto the mode of fluid entry into the system. Fluid entersvia two routes. Plasma can enter through the anastomoses(Vogel, 1985a) probably resulting in constituentconcentrations comparable to the primary circulation.However, the second mode of entry, as an ultrafiltrate fromthe extracellular space (Holmes and Donaldson, 1970), is anindirect path from the primary circulation. Thisultrafiltrate would have a lower concentration of large51molecules, such as protein, and would result in a dilutionof the secondary plasma constituents (Fig. 2.2). However,ions, such as chloride (Fig. 2.2), would not besignificantly lower since they travel easily throughout thebody. Therefore, it is likely that the observed values wereindeed representative of secondary plasma concentrations.A quick decrease in sample sizes occurred as a resultof either clotting within the secondary circulation ordeath of the experimental animals. The clotting may havebeen caused by fibrinogen, which may be present within thesecondary fluid (Holmes and Donaldson, 1970). The largenumbers of thrombocytes in the secondary system (Table 2.1)may be another reason for the high frequency of clotting,since those cells are responsible for clotting mechanisms(Ellis, 1977). The high incidence of death among theexperimental animals implies that they may not haverecovered from the operation. The fluctuating results inthe later sampling periods were produced as a direct resultof individual variation from the remaining fish.A decrease in hematocrit as observed in bothcirculatory systems is typical of animals that arerepeatedly sampled without replacement of blood cells;however, it is also possible that the fish were bleedingfrom both catheters' sites of insertion (Wells and Weber,1991). Vogel (1985a) stated that in an unstressed fish,hematocrit levels within the secondary circulation should be52greater than 1-2 % packed cell volume. However, in thisexperiment, most secondary system samples containedhematocrit levels which were below this value. Thissuggests either that normal hematocrit levels within thesecondary circulation are low at all times, or that the fishwere greatly stressed. The latter explanation was supportedby the high incidence of death among the animals.Hematocrit values are a direct result of the presence of redblood cells. Burne (1926) concluded that there were low,but fluctuating quantities of red blood cells within thesecondary vessels of the Angler fish (Lophius piscatorius).It has been proposed that the secondary vessel network is anutrient vessel system providing the metabolically activeskin with nutrients (Steffensen et al., 1986) such asglucose, and also with hormones (Vogel, 1985a). One of thepurposes of red blood cells is to act as a carrier forhemoglobin, a respiratory pigment, required for oxygentransport (Eckert et al., 1988). Their presence in thesecondary vessels may be to some extent redundant becausethe skin is capable of obtaining its own oxygen throughcutaneous respiration (Krogh, 1904; Privolnev, 1945; Kirscheand Nonnotte, 1977; Nonnotte, 1981; Steffensen and Lomholt,1985; Vogel, 1985a; Takeda 1990). Furthermore, in an oxygenpoor environment it would be extremely costly to bring redblood cells and hemoglobin near the surface thereby losingthe oxygen to the surrounding deficient environment(Steffensen et al., 1982). The significantly lower red53blood cell numbers and hematocrit observed in the secondarysystem in the present study, suggests that the system may beimportant in nutrient, but not in oxygen transport.If the secondary circulation is a nutritive system, itmay be another explanation for the lower concentrations ofcertain variables. Sampling occurred from the lateralcutaneous vessel, a collecting duct close to the terminationof the secondary system (Kampmeier, 1969). Even ifstatistically not significant, there may be biologicalsignificance in decreasing nutrient concentrationsthroughout the secondary circulation as a result ofconsumption by metabolically active cells. Therefore therewould be a lower trend in nutrient concentrations, such asglucose (Fig. 2.2, 2.4), sampled from the lateral cutaneousvessel than in the primary circulation.The low fluid pressure and flow within the secondarycirculation results in a delayed appearance of plasmaparameters compared to the primary system. During stress,cortisol is released into the primary circulation from theinterrenal tissue (Donaldson, 1981; Wedemeyer et al., 1990).At time B (48 hours after surgery), there was aninsignificant increase in primary cortisol concentration(17.8 ± 3.6 gg/dL; time A (24 hours after operation) was12.3 ± 2.3 gg/dL) which is observed in the secondarycirculation approximately 2 hours later at time C (10.6 ±5.9 gg/dL; time A was 5.9 ± 1.8 gg/dL) (Fig. 2.1).54Catecholamine levels can reach concentrations of 10 -6 M in astressed fish (Mazeaud and Mazeaud, 1981). Unfortunately,the disappointing results from the adrenaline analysis (seeTable 2.2) did not provide enough information to determineif the presence of this hormone in the secondary systemlagged behind the primary circulation. The delayed increaseof cortisol concentration suggests that entry into andtravel through the secondary system is slower than for theprimary circulation.Inorganic constituents are similar within the twosystems (Holmes and Donaldson, 1970). If these weresaltwater animals, the secondary system could be importantfor transporting excess chloride to the skin for cutaneouschloride exchange as in the shanny (Blennius pholis L.)(Nonnotte et al., 1979). However, the experimental animalsin this study were freshwater trout; therefore, the slightlylower trend of plasma chloride concentrations observed inthe secondary system (Fig. 2.2, 2.5) may be a result of thision escaping into the chloride poor environment.White blood cell quantities were greater and theircomposition more varied within the secondary circulationcompared with the primary system (Table 2.1). Thisobservation is in contrast to those determined by Wardle(1971) and Ellis and de Sousa (1974) who found similar andlower quantities of white blood cells, respectively, withinthe secondary circulation. On the other hand, Burne (1926)55observed a variation in the number of white and red bloodcells present within sections of "fine" vessels from theAngler fish. He noted areas containing red blood cells andno leucocytes and other locations where the red blood cellswere greatly outnumbered by white blood cells, a 1:5.3 ratiodetermined from a smear of the main abdominal 'lymphatic'.It is plausible that the number of cells vary throughout thesecondary system and that time and length of sampling willresult in the observation of different cell compositions.As summarized by Ellis (1977), thrombocytes come inseveral forms: lone nucleus, oval, spiked and spindle forms.He suggested that slides containing a large numbers of lonenucleus or oval thrombocyte forms be excluded whenperforming differential thrombocyte/lymphocyte cell counts,as these forms are difficult to distinguish from smalldistorted lymphocytes. It is likely that the unknown cellsrepresent a mixture of these cells (Table 2.1) since onlyspiked and spindle forms were counted as thrombocytes.The presence of large numbers of broken cells in thesecondary blood smears made the slides difficult to read.It is unlikely that the staining procedure caused cellbreakage as no visible damage occurred to cells from theprimary circulation. Therefore, it is possible that eitherthe cells ruptured when they came into contact with thecatheter, or that the system contains cell debris suggestinga function similar to that of a mammalian lymphatic system56(Shields, 1972).This experiment characterized the presence of selectedvariables within the secondary circulation. Furtherinvestigation is required to confirm these observations andto clarify the role of the secondary circulatory systemwithin the teleosts.CHAPTER 3:INVESTIGATION OF SELECTED VARIABLES FROM THE PRIMARY ANDSECONDARY CIRCULATORY SYSTEMS IN SIX RAINBOW TROUT(Oncorhynchus mykiss) VACCINATED WITH Vibrio anguillarum.5758INTRODUCTIONFish have an advanced immune system that iscomparable to other "higher" vertebrates. The teleostsecondary circulation may be involved in the immune responsein a similar manner as the mammalian lymphatic system.However, unlike the mammalian lymphatic system, thesecondary circulation does not appear to communicate withthe lymphatic organs (Allen, 1906; Corbel, 1975). Resultingfrom a lack of published material on the secondary system,nothing is known regarding the involvement of thiscirculatory system in the teleost immune response.Teleost fish have several mechanisms which protect themagainst pathogens. Primary barriers, such as mucous, skinand scales, decrease pathogen access to the body (Fletcher,1981). When there is no physical damage to the primarybarriers, the gills are the most important area for pathogenuptake (Anderson et al., 1984). It has been reported thatantigens may also enter via the lateral line (Amend andFender, 1976). Entry into the fish exposes the pathogen toa complex immune system.The teleost immune response is composed of innate andadaptive mechanisms (Playfair, 1984) that are highlydependent on temperature, nutrition and behavior (Corbel,1975). The innate immune system is characterized bycirculating phagocytic cells and stationary macrophages or59dendritic cells that are capable of engulfing foreignmaterial (Corbel, 1975; Anderson et al., 1984). There isalso a humoral component that includes interferon (againstviruses) and lysozyme (Corbel, 1975; Playfair,1984).Lysozyme is an antibacterial enzyme (Mock and Peters, 1990)that hydrolyzes the peptidoglycan in bacterial cell walls(Yousif et al., 1991). Antibacterial compounds which mainlyinhibit the growth of gram negative bacteria, are found onthe skin, in mucous, eye washings and eye extracts fromrainbow trout (Austin and McIntosh, 1988). As well as theseinnate mechanisms, there is also an adaptive immuneresponse.The adaptive immune response is specific in its actionsand results in antigen memory (Corbel, 1975; Playfair, 1984;Roitt, 1988). Briefly, after ingestion and processing,identifying parts that are specific to the pathogen areexposed at the surface of the macrophage and presented tolymphocytes (Playfair, 1984). With the aid of Tlymphocytes, B lymphocytes are stimulated to produce vastquantities of antibodies specific to the antigen (Playfair,1984; Sell, 1987; Roitt, 1988). Memory occurs when some ofthese B, as well as some T cells become dormant; activationoccurs during subsequent exposure to the antigen (Roitt,1988). Unlike mammals, teleosts probably do not have morethan one main immunoglobulin class in their serum (Corbel,1975). Antibodies bind to the surface increasing60phagocytosis of the antigen and also causing neutralizationof bacterial toxins (Playfair, 1984). Complement isinvolved with both the innate and adaptive immune responses.Fish have both a classical and alternate complementpathway (Matsuyama et al., 1988). According to Playfair(1984), Frank (1985), Male (1988) and Roitt (1988)complement can be summarized as follows. It is a series ofserum components resulting in a) inflammation, b)opsonization of the antigen and c) lysis of the antigen.The complement cascade may be initiated by two pathways.The classical pathway is triggered by an antigen-antibodycomplex and involves the adaptive immune response. Thecomplement cascade occurring in the alternate pathway doesnot require an antigen-antibody complex (Matsuyama et al.,1988). Most pathogenic fish bacteria are gram-negative(Ellis, 1988) and the presence of complement aids lysozymein attack against these bacteria (Playfair, 1984). If thepathogen is not completely overwhelmed by the teleosts'immune mechanisms, disease will occur.It is not clear if the teleost secondary circulation isinvolved with the immune system. The mammalian lymphaticsystem is responsible for removing cell wastes, allowing therecirculation of lymphocytes, as well as transportingforeign material such as antigens from the periphery to thelymph nodes (Playfair, 1984; Olszewski, 1985). Althoughfish do not have lymph nodes (Corbel, 1975; Sell, 1987), it61is possible that the secondary circulation has a similarfunction. Ellis and de Sousa (1974) showed that leucocytessampled from the neural duct of plaice (Pleuronectesplatessa), radiolabelled and reinjected into the renalportal vein, are capable of migrating to specific lymphoidorgans; however, the involvement of the secondarycirculation was not concluded.Unlike the mammalian lymphatic vasculature (Olszewski,1985), the secondary circulation in fish does not appear todirectly communicate with the lymphoid organs. For example,in Scorpaenicthys, there is no vascular connection betweenthe lymphomyeloid tissue of the kidney and a secondarycirculatory sinus located directly beneath this organ(Allen, 1906). The normal teleost thymus, located in thegill pouch also does not appear to have connections with thesecondary circulation (Corbel, 1975). An exception to thisis the angler fish (Lophius pisatorius), whose thymus isenveloped by a "lymphatic" sinus directly connected to thesecondary vasculature (Burne, 1926). Communication betweenthe lymphoid organs and the secondary circulatory system isnot common in teleosts.It is not known what role, if any, the secondarycirculation plays in the teleost immune system. Thefollowing experiment used selected immunological techniquesto investigate differences in the immune responses of theprimary and secondary circulatory systems in rainbow trout.62MATERIALS AND METHODS FISHSix rainbow trout, weighing 826.4 ± 52.4 g (mean ± 1s.e.), were used. The fish were obtained from the sametrout farm and maintained in the same manner as those inChapter 1: materials and methods. Water temperature rangedbetween 8 to 9.5 °C.CANNULATIONSThe fish were cannulated in the dorsal aorta followingthe procedure mentioned in Chapter 2: Materials andMethods. The cannulation for the secondary system was themodified method described in Chapter 1: Materials andMethods (part 2, the modified method).EXPERIMENTAL PROCEDURE AND SAMPLINGIn the end of June 1991,^the animals were givenintraperitoneal injections of 0.5 mL mixture of formalinkilled Vibrio anguillarum and V. ordalli cells (Vibrogen-2,Aquahealth Ltd., Canada). No control fish (animals injectedwith saline instead of the vaccine) were included in thisstudy. Vibrio is a gram negative bacteria usually occurringin salt water (Colwell and Grimes, 1984); therefore, it wasassumed that these freshwater trout were naive to thispathogen. Thirteen days post innoculation, three fish werefitted with catheters in both the primary and secondarycirculatory systems. Daily samples (sample A was 24 hours63after surgery; sample B was 48 hours after surgery; sample Cwas 72 hours after surgery; sample D was 96 hours aftersurgery) were obtained until both catheters stopped flowing,or the animals had died. For the last three fish, theoperation was performed on the seventeenth day postvaccination and the sampling sequence initiated thefollowing day. Samples were obtained from the cathetersafter they had been threaded through a hole in the lid ofthe gangbox. At each sampling period, hematocrit wasmeasured, and a blood smear was made for differential cellcounts. Samples were collected by dripping the fluids into4 mL unsealed vaccutainers coated with sodium heparin(Becton Dickenson). Enough fluid was collected to yield 300gL of plasma after centrifugation.Samples were spun at 800 g for 10 minutes (BeckmanModel TJ6, Beckman Instruments, Palo Alto, Ca, U.S.A.) afterwhich the plasma was removed, divided into three 100 ALaliqouts and stored in Eppindorfs at -80 °C until analyzed.The pelleted cells were resuspended with phosphate bufferedsaline (PBS) and processed for use in the plaque assay.PLAQUE ASSAYThe plaque assay quantifies the number of antibodyproducing cells within a sample (Cunningham and Szenberg,1968; Maule et al., 1987, Salonius, 1991). On the day ofsampling, the resuspended cells from the primary circulation64samples were placed onto sterile histopaque (Sigma) andcentrifuged at 800 g for 30 minutes at 16°C to separate thered from the white blood cells. This procedure was omittedfor the secondary samples, because of low red blood cellnumbers. As many white blood cells (WBC) were removed fromthe interface layer of the histopaque as possible. Thecells were placed into a 1.5 mL Eppindorf tube andresuspended with tissue culture medium (TCM, see appendix Bfor composition). Cells from both systems were centrifuged,the supernatant removed and the cells resuspended in 750 ALTCM. Quantification of the number of WBC per mL wasperformed by combining 25 AL of the resuspended cells, 25 ALtrypan blue (0.4% in PBS) and 50 AL of PBS. This mixtureallows the number of viable cells (those that exclude thedye) to be counted when examined using a hemocytometer.Calculation of cell numbers was accomplished using thefollowing formula: # WBC/mL = (# cells counted in 16squares) X 4 (dilution factor) X 10 4 .The plaque assay used in this experiment, quantifiedthe number of antibody producing cells (APC) present withina lawn of Vibrio conjugated sheep red blood cells (V-SRBC).One plaque (an area of lysis within the V-SRBC lawn) wasassumed to equal one APC. Fifty AL resuspended cells, 10 ALsalmon complement (a 1 to 10 dilution of complement andModified Barbital Buffer (MBB), see appendix B forcomposition) and 10 AL of V-SRBC were combined in a wellfrom a 96-well microtitre plate (Falcon). See Appendix B65for complement and SRBC details. The solution was thenplaced into a Cunningham chamber (Cunningham and Szenberg,1968), the chamber sealed with paraffin wax, and incubatedat 17 °C for two hours. Dark field microscopy (25X) wasused to count the number of APC within each chamber. Theresults were quantified as # APC/10 6 viable WBC.PLASMA ANTIBODY TITERSUsing Stolen et al. (1990) as a reference, an antibodytiter test was performed on the plasma. Briefly, 25 gL ofPBS was placed into all wells in a 96-well microtitre plate.Twenty-five pL of previously frozen plasma was depositedinto the first well (one row per sample). After completemixing, 25 gL was removed and placed into the next well inthe same row. This 2-fold dilution was repeated until thesecond from the last well, the last well being used as anegative control. A similar dilution was duplicated using apositive control of known antiserum. Fifty pL of Vibriocells were then added to each well. Each plate was covered,incubated at 17 °C for approximately 18 hours and thenexamined. The most dilute point where agglutinationoccurred was determined as the antibody titer.PLASMA LYSOZYMELysozyme concentration was determined by placing 10 gLof previously frozen plasma into wells punctured in agarosegel plates containing Micrococcus lysodeikticus ( freeze66dried, Sigma). Similar results are obtained by using eitherplasma or serum in this assay (Mock and Peters, 1990). Theplates were incubated at 20-25°C within a moist chamber for17 hours. The gram positive bacteria is sensitive to thepresence of lysozyme; the diameter of lysis (clearance zone)surrounding the wells being directly proportional to theconcentration of plasma lysozyme. Log regression analysisusing the following formula was used to determine thelysozyme activity: Y (diameter of lysis) = A + B•logX(lysozyme activity, units/mL). Salonius (1991) and Yousifet al. (1991) were used as references for this technique.HEMATOLOGYHematocrit and differential white blood cell countswere determined using the techniques specified in Chapter 2:Materials and Methods.PLASMA CORTISOLCortisol concentration was measured following theradioimmunoassay described in Chapter 2: Materials andMethods.TOTAL PLASMA PROTEINThe analysis of total protein was performed using themethod outlined in Chapter 2: Materials and Methods.67PLASMA GLUCOSEPlasma glucose concentration was measured using themodified ortho-toluidine method explained in Chapter 2:Materials and Methods.PLASMA CHLORIDEChloride concentration was determined following thetechnique described in Chapter 2: Materials and Methods.STATISTICAL TREATMENTResults were subjected to the same statistical analysisas the data from Chapter 2: Materials and Methods.68RESULTSSampling took 5.0 ± 0.8 and 32.5 ± 5.9 minutes for theprimary and secondary, respectively. Unfortunately, plaqueassay results were poor with plaques occurring in less thanhalf of all samples (Table 3.1). The appearance of generallysis throughout the V-SRBC lawn in many secondarycirculatory samples was not attributed to antibody producingcells. There was a negative response by all samples to theantibody titer test. The positive response from theantiserum control indicated that these results were notartifacts.Although plasma lysozyme activity was not statisticallydifferent between the two groups, there was a higher trendin the secondary than the primary concentration (Fig. 3.1).Lysozyme in the secondary circulation was calculated at114.9 ± 14.9% of the primary concentration (Fig. 3.2). Nocorrelation was found between lysozyme activity and totalleucocyte numbers.Hematocrit values were less than 1% packed cell volumein the secondary system, resulting in a highly significantdifference between the two groups (Table 3.2). The primarysystem showed a general decrease in hematocrit over the foursampling periods. As in Chapter 2, cell composition variedgreatly in the secondary circulation. The leukocyteconcentration was higher within the secondary system while69red blood cells quantities were lower than the primarycirculation. These results were statistically different.Plasma cortisol concentration was not significantlydifferent between the two groups (Fig. 3.3). The cortisolconcentration of the secondary system was generally lessthan the primary circulation, and was calculated at 51.4 ±17.2% (Fig. 3.2).The concentration of total protein was significantlygreater in the primary than the secondary circulation (Fig.3.4). Further analysis revealed that the difference was attime C (P<0.05). The protein concentration of the secondarysystem was determined to be 71.1 ± 7.5% of the primarycirculation (Fig. 3.2).Both the primary and secondary circulation glucoseconcentrations decreased during the sampling periods (Fig.3.5). Although there was no difference between groups, thegroups themselves contained means that were significantlydifferent from each other (primary circulation between timesA and D; secondary between A and C). The glucoseconcentration of the secondary circulation was determined tobe 92.1 ± 11.8% of the primary circulatory concentration(Fig. 3.2).Plasma chloride concentration was similar in bothsystems as was the trend that they followed (Fig 3.6).Generally, chloride concentration in the secondary70circulation was slightly lower resulting in the chloridefrom the secondary system being 96.4 ± 2.3% of the primarycirculation (Fig. 3.2).71SAMPLE SIZE(n)PLAQS PLAQSCALC.PRIMARY CIRCULATIONA 1 2.0 30.0(6) - -B 3 14.7 191.7(5) 1.5 16.9C 1 1.0 15.0(4) - -D 2 4.0 60.0(2) 3.0 45.0SECONDARY CIRCULATIONA 1 9.0 90.0(5)B 2 2.5 37.5(4) 1.5 22.5C 2 2.5 37.5(2) 0.5 7.5D 1 6.0 90.0(1)WBC WBC PLAQS/CALC. WBC222.5^8.9^7.3293 1.2 -339.0^13.6^61.7107.5 4.3 40.4425.3^17.0^0.4153.7 6.1889.0^35.6^1.79.0 0.4 1.3422.1^16.9^45.5128.6 5.184.8^3.4^21.662.0 2.5 20.3627.5^25.1^6.1566.6^22.7 5.6158.0^6.3^14.2Table 3.1 ) Means ± 1 s.e. of the hemolytic plaque assayand live white blood cell counts for six rainbow troutvaccinated two weeks earlier with formalin killed Vibrioanguillarum. Numbers in brackets are the sample sizes fromthe WBC counts while the other sample sizes are from theplaque assay.Plaqs = number of counted plaques; Plaqs caic. = calculatednumber of plaque forming cells within the sample; WBC =white blood cells; WBC caic. = calculated number of WBCwithin the sample ;  = number of plaques per 10 °WBC. * WBC x 106'. + these figures only include WBC fromsamples containing plaques.Time A = 1 day after surgery; B = 2 days after surgery; C =3 days after surgery; D = 4 days after surgery.72Table 3.2) Cell counts and hematocrit (mean ± 1 s.e.) fromthe primary and secondary circulation in rainbow troutvaccinated two weeks earlier with Vibrio anguillarum.Statistics was only performed on the raw data. There was nosignificant difference within each group. A significantdifference was found between the overall means thrombocytesand neutrophils (P<0.05); unknown cells, red blood cells andhematocrit (P<0.001). Paired t-tests between the twocirculatory systems also revealed diffqrences at theindividual sampling periods: a = P<0.001, " = P<0.01, z =insufficient data. * Total WBC do not include unknown cellsin the primary circulation.neutro = neutrophil; throm = thrombocyte; lymph =lymphocyte; unknown = cells that could not be categorized,but were probably thrombocytes and/or small distortedlymphocytes; WBC = white blood cells; RBC = red blood cells;HCT = hematocrit; slides that contained zero RBC were notincluded in the standardized cell counts, numbers inbrackets reflect the smaller sample sizes. See Table 3.1for description of sample times.AVERAGE CELL COUNTS CELL COUNTS AS PERCENTAGE OF TOTALCELLSSample^Size(n)Neutro^Throm Lymph Un-knownTotalWBCTotalRBCNeutro Throm Lymph Un-knownRBC HCTPRIMARY CIRCULATIONA^6 0.5 0.2 5.2 0.0z 1.9 737.0a 0.07 0.02 0.7 0.0 99.2 26.2z0.2 0.2 0.8 0.6 61.1 0.03 0.02 0.07 0.0 0.1 1.3B^5 0.2 0.2 7.8 0.0z 2.7 B 0.02 0.03 1.0 0.0 98.9 30.0z0.2 0.2 3.1 1.4 0.02 0.03 0.4 0.0 0.4 3.1C^4 0.8z 0.3z 7.3z 0.V 2.8 827.8z 0.1 0.03 0.8 0.0 99.1 24.6z0.3 0.3 3.0 1.3 109.7 0.03 0.03 0.2 0.0 0.2 2.5D^2 0.0z 0.0z '7.0z 0.V 2.3 688.0z 0.0 0.0 1.0 0.0 99.0 21.0z0.0 1.5 50.0 0.0 0.0 0.07 0.0 0.07 4.0SECONDARY CIRCULATIONA 4 18.0 19.4 2.9 48.5 22.2 16.5 16.7 16.0 3.0 43.3 21.0 <110.0 6.9 1.8 14.7 6.1 7.5 7.2 4.8 1.6 11.9 12.9B 3 9.3 1.0 18.0 29.3 14.4 86.7 7.8 0.6 9.6 19.8 62.2 <15.6 0.6 12.8 23.6 6.6 64.8 3.5 0.4 2.9 17.7 20.4C 1 7.0 5.0 1.0 71.0 21.0 17.0 6.9 5.0 1.0 70.3 16.8 <116.7D 0\1//11400—Primary circulationSecondary circulation741200J--1Ecr,D'-'-' 100051---U< 8001J..1>"--N0(f)600<Co)<_ILL 400200A^B^C^DSAMPLE PERIODSFigure 3.1)^Means ± 1 s.e. of plasma lysozyme activity(Ag/mL) from the primary and secondary circulation inrainbow trout vaccinated two weeks earlier with formalinkilled Vibrio anguillarum. Numbers indicate sample sizes.Statistical analysis revealed no significant differencesbetween or within the two circulatory systems. See Table3.1 for description of sample times.130 -1201101 00z0r=< 80_JDo 70U>- 60<500_L 4003020100lir^'V0 40^4■ 40^4I 40^40 4I^40^10 40^4I 4■^4I 4I^40 4I^40 4■^4I 40^4■ 40^4I 40^40 ILA*. I90 *75Co rtiso Glucose^total^Chloride LysozymeproteinSELECTED SECONDARY PARAMETERSFigure 3.2)^Selected secondary parameters expressed aspercent of primary circulation.^* indicates a statisticaldifference between the raw data from the two circulatorysystems (P<0.01).CB DA1 0Primary circulationSecondary circulation.^.1//182SAMPLE PERIODSFigure 3.3) Means ± 1 s.e. of plasma cortisol (pg/dL) fromthe primary and secondary circulation in rainbow troutvaccinated two weeks earlier with formalin killed Vibrioanguillarum. Numbers indicate sample sizes. Statisticalanalysis revealed no significant differences between orwithin the two circulatory systems. See Table 3.1 fordescription of sample times.76//Primary circulationSecondary circulationreA1 077A^DSAMPLE PERIODSFigure 3.4) Means ± 1 s.e. of total protein (g/dL) from theprimary and secondary circulation in rainbow troutvaccinated two weeks earlier with formalin killed Vibrioanguillarum. Numbers indicate sample sizes. The two groupswere statistically different from each other (P<0.01).Further analysis did not disclose at which sampling periodthe difference occurred. See Table 3.1 for description ofsample times.78200ab^c175150125 a400755025Primary circulationSecondary circulationbA^B^C^DSAMPLE PERIODSFigure 3.5) Means ± 1 s.e. of plasma glucose (mg/mL) fromthe primary and secondary circulation in rainbow troutvaccinated two weeks earlier with formalin killed Vibrioanguillarum. Numbers indicate sample sizes. Letters thatare the same are statistically different within each group(P<0.05). See Table 3.1 for description of sample times.140 —79M Primary circulationSecondary circulation1352130o-1250(r) 120_J115110A DSAMPLE PERIODSFigure 3.6) Means ± 1 s.e. of plasma chloride (mEq/L) fromthe primary and secondary circulation in rainbow troutvaccinated two weeks earlier with formalin killed Vibrioanguillarum. Numbers indicate sample sizes. Statisticalanalysis revealed no significant differences between orwithin the two circulatory systems. See Table 3.1 fordescription of sample times.DISCUSSIONAs in Chapter 2, the trends of plasma constituents suchas cortisol, protein, glucose and chloride were lower in thesecondary system than the primary circulation; however, onlytotal protein was significantly different (see Figures 3.2,3.3, 3.4, 3.5 and 3.6). The hematocrit was also lower andthe leucocyte numbers varied much more within the secondarycirculation. The large quantities and varying compositionof the white blood cells at the different sampling periodsmay have resulted from an inflammatory response (Finn andNielson, 1973).Vibriosis vaccines are highly effective in protectingsalmonids against Vibrio outbreaks (Ellis, 1988). Onlysmall amounts of vaccine are required to offer protectionwhen given by intraperitoneal injection (Thorburn et al.,1989; Velji et al., 1990). Therefore, the 0.5 mL of vaccineinjected into the rainbow trout in this experiment shouldhave been adequate to stimulate an immune response.The disappointing plaque assay results, however, didnot permit the conclusion of the secondary circulations'involvement in the adaptive immune response. It is unlikelythat the complement source was inactive, as some Cunninghamchambers did contain plaques. However, inexperience usingthis assay may have resulted in the misreading of the8081slides. Another possible explanation is that the samplingperiod missed the correct window for examining antibodyproducing cells. However, Sakai et al. (1984) determinedthat the maximum number of plaques occurred 13 days postvaccination in rainbow trout. Kaattari and Irwin (1985)found that the spleen and anterior kidney contained maximumplaque forming cells on day 16 in coho salmon (Oncorhynchuskisutch). Maule et al. (1987) examined coho salmon 17 daysafter injection of Vibrio. Therefore, the sampling periodfor this experiment, starting on day 13 post vaccination,corresponded with the sampling periods from otherresearchers.The antibody titer test was negative from all fishduring the experiment. This humoral component of theadaptive immune response is produced by antibody producingcells (Playfair, 1984). Therefore, cell mediated responsesprecede antibody production (Groberg et al, 1982; Blazer etal., 1984). Antigen presenting cells appear one week afterantigen encounter, peaking on day 14 in splenic cells, whilethe antibody production starts on day 15 (Anderson et al.,1984). Sakai et al. (1984) determined that antibodyproduction started on day 13 and peaked on day 28 aftervaccination in rainbow trout. These time scales indicatethat antibody titers should have been present within thesamples. Using a more sensitive technique, such as an ELISAassay, may have detected any low levels of antibody from the82experimental animals (Thuvander et al, 1987; Thorburn etal., 1989).There are several possible explanations for the lowplaque numbers and negative antibody titers. Unlikehomeothermic mammals, the fish immune system is highlydependant on environmental temperature (Corbel, 1975;Kaattari and Irwin, 1985). In particular, low temperaturesaffect the classical complement pathway thereby decreasingantibody production; however, phagocytosis and the alternatecomplement pathway are not inhibited at lower temperatures(Fletcher, 1986; Matsuyama et al., 1988). The temperaturein this experiment ranged between 8 and 9.5 °C and may havebeen low enough to slow the immune response (Groberg et al.,1983).The physiological state of the fish is closely relatedto the ability of producing an immune response (Groberg,1983; Anderson et al., 1984). For example, stress causeslymphopenia (McLeay, 1975). An increase in plasma cortisol,a stress hormone, corresponds with a decrease in survival ofsmolts exposed to Vibrio (Maule et al., 1987). Cortisol isalso found to significantly reduce the migration ofperitoneal neutrophils in plaice (Fletcher, 1986).Glucocorticoids interfere with macrophage receptors reducingtheir ability to present antigen and thereby resulting in adecrease in an antibody response (Frank, 1985). The traumafrom the operation may have affected the immune response of83the fish in this study. Nutritional factors also affect theanimals' ability to react to antigens (Corbel, 1975). Thedecrease in plasma glucose (Fig. 3.4) suggests that duringthe sampling period, there was a reduction in availableenergy for the fish.When animals first encounter an antigen, there is onlya small response from the adaptive immune system; however,on subsequent exposures, the cellular response is not onlygreater, but also much faster. For example, Blazer et al.(1984) found that a second innoculation produced maximumquantities of plaque forming cells on day 6 (133 PFC/10 6WBC) compared with day 14 (100PFC/10 6 WBC) after the firstvaccination. The single exposure of the fish to the Vibriovaccine in this experiment, may have resulted in a smalladaptive immune response.Even though plasma lysozyme activity within thesecondary circulation was not statistically different fromthe primary system, the trend of higher activity within thesecondary circulation may be the result of the small samplesize and not biological reality (Figures 3.1, 3.2). Agreater concentration within the secondary circulationcannot be concluded as being the norm since there was nocomparable data for control fish. However, the activity oflysozyme does vary at different locations within the body.Mock and Peters (1990) determined that the activity withinthe kidney is a 10 to 20X greater than in other parts of the84fish. The presence of lysozyme cannot be attributed only tothe vaccination as this enzyme is not specific in itsactions. There are two plausible explanations for greaterlysozyme activity within the secondary system. Thesecondary system may be a storage facility for humoralcomponents of the innate immune function. These are thenreadily available to attack antigens that enter via theskin. Alternately, the secondary system, like the mammalianlymphatic system, may transport cell wastes, includingexcess lysozyme (Roitt, 1988). Lysozyme activity can bedecreased by a strong stressor (such as an operation) (Mockand Peters, 1990). The lack of a standardized technique forthis assay results in difficulties when comparing data fromother researchers (Mock and Peters, 1990).It should be noted that there was no adjuvant includedin the vaccine administered during this experiment.Adjuvants are immunopotentiators that augment the innate andadaptive immune responses (Roitt, 1988). They are oftenincluded during vaccination to stimulate non-specificdefense mechanisms (activation of macrophages) therebyincreasing the range of pathogens against which the fishwill be protected (Ellis, 1988). For example, Freund'scomplete adjuvant (FCA) was used by Sakai et al. (1984);therefore, their results may not be characteristic of anotherwise unstimulated immune response.The aim of this experiment was to compare the immune85response within the primary secondary circulatory systems.The disappointing results for both the plaque assay andantibody titer test do not conclude the activity of thesecondary circulation in the adaptive immune response. Itis possible that the conditions were not ideal; therefore,the adaptive immune response was not stimulated. The highertrend in lysozyme concentration suggests that the secondarycirculation is important in the innate immune function,possibly acting as a reservoir for humoral products. Thepossible involvement of the secondary circulation in theteleost immune response requires further study.CHAPTER 4:86SUMMARY AND RECOMMENDATIONS87A new cannulation technique of the secondarycirculation was described in Chapter 1 and used for theexperiments in Chapters 2 and 3. This author knows of noother published method for the chronic cannulation of thesecondary circulation in teleosts. The use of an indwellingcatheter is advantageous for two reasons: (1) multiplesamples may be taken from the same individual and (2) thefish is removed from the experimenter, resulting in measuredvariables that are more representative of an unstressed fishcompared with experimental animals that are constantlyhandled during sampling. However, the high mortality of thefish used in the experiments suggests that they may not haverecovered from the surgery. The described technique isassociated with problems (such as clotting) resulting fromthe nature of this circulatory system. In fact, clottingand fish mortalities produced a reduction of the sample sizeby approximately 60% after the second sampling period. Itis therefore recommended that the method be further examinedand improved upon to increase its reliability.Selected variables from both the primary and secondarycirculatory systems of rainbow trout were characterized inChapter 2. By using the described cannulating technique,concentrations of plasma cortisol, adrenaline, totalprotein, glucose, chloride and also hematocrit anddifferential cell counts were determined for both systems.Generally, the secondary system showed a lower trend for the88constituents than the primary system in all measuredvariables except white blood cells. White blood cellnumbers were highly variable indicating that cell contentwithin the secondary system continually changes. As aresult of the low pressures and larger volume, the secondarysystem appears to have a slower mixing time than the primarycirculation. This was concluded from an increase in plasmacortisol concentration in the primary system that was laterobserved in the secondary circulation by a similar increase.The small quantities of red blood cells in the secondarysystem suggest that this circulatory system is not importantfor oxygen transport. Decreasing the time required tosample this system, would allow for the analysis of otherplasma constituents (eg. pH, blood gases) that are regularlyinvestigated in the primary circulation. It would bevaluable to continuously monitor physical parameters, suchas flow and pressure, to further characterize the secondarycirculation.The study in Chapter 3 examined the immune response ofboth the primary and the secondary systems in rainbow troutto Vibrio anguillarum. As was determined in the previousexperiment, there was a lower trend for most of the measuredvariables in the secondary circulation. White blood cellnumbers were significantly greater and varied more thanthose in the primary circulation. Although not significant,lysozyme concentration appeared higher within the secondary89circulation. This finding indicates that the secondarycirculation may be important for the innate immune response.Its large volume and extensive vessels system may provide areservoir for immune products enabling them to react quicklyto antigens entering the body via the skin. The low numberof samples responding to the hemolytic plaque assay andnegative response of all samples in the antibody titer testsuggest that the specific immune response was notstimulated. It is possible that the environmentalconditions were not ideal; therefore, the specific humoralresponse was not observed. This experiment does notconclude an involvement of the secondary system in theadaptive immune response. It is therefore recommended thatthe investigation be repeated using a larger sample size andincluding control fish. Re-vaccination of the animals a fewdays prior to operation would also result in a more visibleimmune response.This study has broken new grounds in the study of thesecondary circulation in rainbow trout. The use ofindwelling catheters resulted in selected primary andsecondary circulatory parameters being characterized. Thesecondary circulation may be important for the innate immuneresponse; however, its' role in the adaptive immune responsewas not surmised. Further investigation is required toascertain the secondary circulations' involvement in theimmune response, and to recognize other possible functions90of this extensive vessel system. It is hoped that thedescribed cannulating techniques and subsequent experimentswill aid the research community in the acquisition of newinformation regarding the secondary circulatory system inteleosts.91REFERENCES Alexander, J.B. and Ingram, G.A. (1980). A comparison offive of the methods used to measure proteinconcentration in fish sera. J. Fish. Biol. 16: 115-122.Allen, W.F. (1906). Distribution of the lymphatics in thehead, and in the dorsal, pectoral and ventral fins ofScorpaenicthys marmoratus. Proceedings of theWashington Academy of Sciences. 8: 41-90.Amend, D.F. and Fender, D.C. (1976). Uptake of bovine serumalbumin by rainbow trout from hyperosmotic solutions:a model for vaccinating fish. Science 192: 793-798.Anderson, D.P. (1974). Fish Immunology: Diseases ofFishes. TFH Publications, Inc. Ltd. Hong Kong.Anderson, D.P., van Muiswinkel, W.B. and Roberson, B.S.(1984). Effects of chemically induced immunemodulation on infectious diseases of fish. ChemicalRegulation of Immunity in Veterinary Medicine. pp. 187-211.Austin, B. and McIntosh, D. (1988). Natural antibacterialcompounds on the surface of rainbow trout, Salmogairdneri Richardson. J. Fish Diseases 11: 275-277.Blazer, V.S., Bennett, R.O. and Wolke, R.E. (1984). Thecellular immune response of rainbow trout (Salmogairdneri Richardson) to sheep red blood cells. Dev.Comp. 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Physiological evidence for theoccurrence of pathways shunting blood away from thesecondary lamellae of eel gills. J. Exp. Biol. 98:277-288.Hewson, W. (1796). An account of the lymphatic system infish. Phil. Trans. R. Soc. 59: 204-215.Holmes, W.N. and Donaldson, E.M. (1970). The bodycompartments and the distribution of electrolytes. In:Fish Physiology. Hoar, W.S. and Randall, D.J. (eds.).1: 1-89.Jourdain, P. M. S. (1868). Le system lymphatique du gadusmorrhua. Annls. Sc. Nat., Ser. Zool. T8; 141-144.Jourdain, P.M.S. (1880). Sur l'existence d'une circulationlymphatique chez les Pleuronectes. Seanc. Acad. Sci90: 1430-1432.Kaattari, S.L. and Irwin, M.J. (1985). Salmonid spleen andanterior kidney harbor populations of lymphocytes withdifferent B cell repertoires. Dev. Comp. Immunology9: 433-444.Kampmeier, O.F. (1969). Evolution and ComparativeMorphology of the Lymphatic System. Charles C. ThomasPublisher, Illinois. Chapters I, VII.Kirsch, R. and Nonnotte, G. (1977). Cutaneous respirationin three freshwater teleosts. Resp. Physiol. 29:339-354.Korcock, D.E., Houston, A.H. and Gray, J.D. (1988). Effectsof sampling conditions on selected blood variables ofrainbow trout, Salmo gairdneri Richardson. J. FishBiol. 33: 319-330.Krogh, A. (1904). Some experiments on the cutaneousrespiration of vertebrate animals. Scand. Arch.Physiol. 16: 348-357.94Krogh, A. (1959a). Lecture I: Introductory - the flow ofblood in the microscopic vessels. In: The anatomy andphysiology of capillaries. Hafner Publishers, NewYork. pp. 1-21.Krogh, A. (1959b). Lecture III: The independentcontractility of capillaries. In: The anatomy andphysiology of capillaries. Hafner Publishers, NewYork. pp. 47-69.Langen, J.K. and McIntyre, P.A. (1981). The hematopoieticsystem. In Nuclear Medicine Technology and Techniques.Pernier, D.R., Langen, J.K. and Wells, L.D. (Eds.).The C.V. Mosby Company, St. Louis. pp. 403-420.Larkin, P.A. (1975). Biology 300 (Biometrics) some notesand problems. University of British Columbia.Lehmann, J. (1988). Farbatlas der Histologie der RegenbogenForelle. Landesanstalt fur Fischerei Nordrhein-Wesfalen. Springer-Verlag, Berlin.Male, D. (1988). Complement. IRL Press, Oxford.Matsuyama, H., Tanaka, K., Nakao, M. and Yano, T. (1988).Characterization of the alternative complement pathwayof carp. Dev. Comp. Immun. 12: 403-408.Maule, A.G., Schreck, C.B. and Kaattari, S.L. (1987).Changes in the immune system of coho salmon(Oncorhynchus kisutch) during the parr-to-smolttransformation and after implantation of cortisol.Can. J. Fish. Aquat. Sci., 44(1): 161-166.Mayer, P. (1917). Uber die lymphgefasse der fisher and ihremUtmassliche rolle bei der verdauung. JenaischeZeitschrift fur Naturwissenschaft 55: 123-174.Mazeaud, M.M and Mazeaud, F. (1981). Adrenergic responsesto stress in fish. In: Stress and Fish. Pickering,A.D. (ed.). Academic Press, New York. pp. 49-183.McLeay, D.J. (1975). Sensitivity of blood cell counts injuvenile coho salmon (Oncorhynchus kisutch) tostressors including sublethal concentrations ofpulpmill effluent and zinc. J. Fish. Res. Board Can.32(12): 2357-2364.Mock, A and Peters, G. (1990). Lysozyme activity in rainbowtrout, Oncorhynchus mykiss, (Walbaum), stressed byhandling, transport and water pollution. J. Fish Biol.37: 873-885.95Nichols, D.J. (1987). Fluid volumes in rainbow trout, Salmogairdneri: application of compartmental analysis.Nilsson, S. (1986). Control of gill blood flow. In: FishPhysiology: recent advances. (Eds.) Nilsson, S.,Holmgren, S. and Croom Helm, S. pp. 86-101.Nonnotte, G. (1981). Cutaneous Respiration in sixfreshwater Teleosts. Comp. Biochem. Physiol. 70A:541-543.Nonnotte, G., Nonnotte, L. and Kirsch, R. (1979). Chloridecells and chloride exchange in the skin of a sea-waterteleost, the shanny (Blennius pholis L.). Cell TissueRes. 199: 387-396.Olson, K.R. (1984). Distribution of flow and plasmaskimming in isolated perfused gills of three teleosts.J. Exp. Biol. 109: 97-108.Olson, K.R. and Kent, B. (1980). The microvasculature ofthe Elasmobranch gill. Cell Tissue Res. 209: 49-63.Olszewski, WL. (1985). Peripheral lymph: formation andimmune function. CRC Press, Inc. Florida, U.S.A.Playfair, J.H. (1984). Immunology at a glance. BlackwellScientific Publications, Oxford.Privolnev, T.I. (1945). Skin respiration in Carassiuscarassius. Comptes rendus (Doklady) de l'Academie dessciences de l'URSS. 48(8): 594-596.Randall, D.J. (1970). The circulatory system. In: FishPhysiology Volume IV. Hoar, W.S. and Randall, D.J.(eds.). Academic Press, New York. pp. 133-172.Redding, J.M., Schreck, C.B., Birks, E.K. and Ewing, R.D.(1984). Cortisol and its effects on plasma thyroidhormone and electrolyte concentrations in fresh waterand during seawater acclimation in yearling cohosalmon, Oncorhynchus kisutch. Gen. Comp. Endocrinol.56: 146-155.Roitt, I. (1988). Essential Immunology. BlackwellScientific Publications, London.Rowing, C.G.M. (1981). Interrelationships between arteries,veins and lymphatics in the head region of the eel,Anguilla anguilla L. Acta Zool. 62(3): 159-170.Runyon, R.P. (1985). Fundamentals of Statistics in theBiological, Medical, and Health Sciences. DuxburyPress, Boston.96Sakai, M., Aoki, T., Kitao, T., Rohovec, J.S. and Fryer,J.L. (1984). comparisons of the cellular immuneresponse of fish vaccinated by immersion and injectionof Vibrio anguillarum. Bull. Jap. Soc. Sci. Fisheries.50(7): 1187-1192.Salonius, K. (1991). Effects of early rearing history onselected endocrine and immune functions in juvenilepacific salmonids. M.Sc thesis, University of BritishColumbia.Sell, S. (1987). Basic immunology: immune mechanisms inhealth and disease. Elsevier Science PublishingCompany, Inc., New York.Shields, J.W. (1972). The Trophic Function of LymphoidElements. Charles Thomas Publisher, Illinois.Smith, L.S. and Bell, G.R. (1964). A technique forprolonged blood sampling in free-swimming salmon. J.Fish. Res. Bd. Canada 21(4): 711-717Soivio, A., Nynolm, K. and Westman, K. (1975). A techniquefor repeated sampling of the blood of individualresting fish. J. exp. Biol. 62: 207-217.Steffensen, J.F. and Lomholt, J.P. (1985). Cutaneous oxygenuptake and its relation to skin blood perfusion andambient salinity in the plaice, Pleuronectes platessa.Comp. Biochem. Physiol. 81A (2): 373-375.Steffensen, J.F., Lomholt, J.P. and Vogel, W.O.P. (1986).In vivo observations on a specialized microvasculature,the primary and secondary vessels in fishes. Acta Zool(Stockh). 67(4): 193-200.Stolen, J.S., Fletcher, T.C., Anderson, D.P., Roberson, B.S.and Muiswinkel, W.B. (1990). Techniques in fishimmunology. SOS Publications, Fair Haven, New Jersey.Takeda, T. (1990). Cutaneous and gill oxygen uptake in thecarp, Cyprinus carpio, as a function of metabolic rate.Comp. Biochem. Physiol. 95A (3): 425-427.Thuvander, A. (1987). Duration of protective immunity andantibody titres measured by ELISA after vaccination ofrainbow trout, Salmo gairdneri Richardson, againstvibriosis. J. Fish Diseases 10: 479-486.Torburn, MA., Jansson, E. and Thuvander, A. (1989).Vibriosis vaccination of rainbow trout Salmo gairdneriat varying temperatures and seasons. II. Effects onantibody production in five Swedish field trials. Dis.97Aquat. Org . 6: 27-32.Velji, M.I., Albright, L.J. and Evelyn, T.P.T. (1990).Protective immunity in juvenile coho salmonOncorhynchus kisutch following immunization with Vibrioordalii lipopolysaccharide or from exposure to live V.ordalii cells. Dis. aquat. Org . 9: 25-29.Vogel, W. 0. P. (1978). The origin of Fromm's artery introut gills. Z. mikrosk. anat. Forsch. 92: 565-570.Vogel, W.O.P. (1981). Struckture and organisationsprinzipim Gefasssystem der knochenfische. Gegenbaurs morph.Jahrb., Leipzig 127. 6: 772-784.Vogel, W.O.P. (1985a). Systemic vascular anastomoses,primary and secondary vessels in fish, and thephylogeny of lymphatics. Cardiovascular Shunts, A.Benzon Symposium 21. (Eds.) Johansen, K. andBurggren, W. W. Munksgaard.Vogel, W.O.P. (1985b). The caudal heart of fish: not alymph heart. Acta. anat. 121: 41-45.Vogel, W.O.P. and Claviez, M. (1981). Vascularspecialization in fish, but no evidence for lymphatics.Z. Naturforsch. 36c: 490-492.Vogel, W.O.P., Vogel, V. and Kremers, H. (1973). Newaspects of the intrafilamental vascular system in gillsof a euryhaline teleost, Tilapia mossambica. Z.Zellforsch. 144: 573-583.Vogel, W.O.P., Vogel, V. and Pfautsch, M. (1976). Arterio-venous anastomoses in rainbow trout gill filaments -- ascanning and transmission electron microscopic study.Cell Tiss. Res. 167: 373-385.Vogel, W.O.P., Vogel, V. and Schlote, W. (1974).Ultrastructural study of arterio-venous anastomoses infill filaments of Tilapia mossambica. Cell Tiss. Res.155: 491-512.Wardle, C.S. (1971). New observations on the lymph systemof the plaice Pleuronectes platessa and other teleosts.J. Mar. Biol. Ass. U.K. 51: 977-990.Wedemeyer, G.A., Barton, B.A. and McKeay, D.J. (1990).Stress and acclimation. In: Schreck, C.B. and Moyle,P.B. (eds.). Methods for Fish Biology. AmericanFisheries Society, Bethesda, Maryland.Wells, R.M.G. and Weber, R.E. (1991). Is there an optimalhematocrit for rainbow trout, Oncorhynchus mykiss98(Walbaum)? An interpretation of recent data based onblood viscosity measurements. J. Fish Biol. 38: 53-65.Wilkinson, L. (1988). SYSTAT: The system for statistics.SYSTAT Inc., Evanston, Illinois.Wolf, K. (1963). Physiological salines for fresh-waterteleosts. The Progressive Fish-Culturist. July: 135-140.Woodward, J.J. (1982). Plasma catecholamines in restingrainbow trout, Salmo gairdneri Richardson, by highpressure liquid chromatography. J. Fish Biol. 21:429-432.Yasutake, W.T. and Wales, J.H. (1983). Microscopic Anatomyof Salmonids: an atlas. United States Department ofthe Interior, Fish and Wildlife Service. ResourcePublication 150, Washington, D.C.Yousif, A.N., Albright, A.L. and Evelyn, T.P.T. (1991). Theoccurrence of lysozyme in the eggs of coho salmonOncorhynchus kisutch. Dis. Aquat. Org . 10: 45-49.99APPENDIX A: Construction of T-pieces for secondary cannulationsThe following is an account of the best method formaking catheters used for long term sampling from thesecondary circulation (lateral cutaneous vessel) ofsalmonids.MATERIALS- Polyethylene tubing (PE-60 from Clay Adams, usedin fish that were >600 grams)- Dissecting microscope- Soldering iron with an unraveled paperclipfastened to the heated surface creating a hotsurface with a very fine tip. The paperclipallows for delicate work and distributesenough heat to the plastic that itbecomes pliable but doesn't melt.- 2 metal guitar strings that fit easily insidethe tubing yet obscure most of the lumen.- a scalpel and new blades- syringe filled with water, and needle tip whichfits snugly into the tubing (21g1 gauge fromBecton Dickenson for PE-60). To preventdamage to the tubing, the sharp needle tip isfiled off.- two clamps.METHODA long piece of tubing is cut approximately 50 cm inlength. This length is not crucial, however, a shorterpiece that is lengthened by a connector (a piece of a hollowneedle that has been filed smooth on both ends) is a majorsource of problems. The connector's smaller lumen oftenprevents clots from passing, thereby stopping the flow. Itis painstaking work to clean out the catheter and get the100Figure A)^Construction of the T-piece for cannulation ofthe lateral cutaneous vessel: 1) Polyethylene tubing (a)with a small notch (N) removed about 3 cm from one end, 2)insertion of a guitar wire (W1) into the tubing and throughthe notch, another wire (W2) is threaded into the rest ofthe tubing, 3) a 3 cm piece of tubing (b) is cut, 4) heatingthe end of piece (b) causes it to melt and flare, 5) theflared end of (b) is quickly inserted onto the protrudingwire (W1) and the plastic wrapped around (a) (W2 is notshown to increase the clarity), 6) the W1 wire is completelyinserted into (b). Under a microscope, the paperclip tip(P) of the soldering iron (S) is used to smooth out theplastic and seal the connection, 7) the final product aftertesting for leakage.bFigure A)11r--w2 -06-..:::::. • _.^.................. ..... ....................- A. . ....... ..... . .)\N101(1 0^102fluid flowing again. For this reason, a long length is moredesirable. 3 cm from the end, the tubing is slightly bentand a small triangle is cut out using a very sharp blade(Fig. A.1). This area is viewed under the dissecting scopeto ensure that it is a clean cut. A guitar wire is insertedinto the end of the tubing closest to the cut and fedthrough the hole till it protrudes about 0.5 cm (Fig. A.2).The other guitar string is then inserted from the other enduntil it juxtaposes the protruding wire at 45 degrees (Fig.A.2).Another piece of tubing is cut to a 3 cm length (Fig.A.3). Using the soldering iron, this piece is heated on oneend causing it to flare (Fig. A.4). This end is immediatelyplaced onto the protruding guitar wire and molded around theother tubing; in this way, the top of the "T" is made (Fig.A.5). The formerly protruding wire is inserted into thenewly attached tube.^The union is viewed using thedissecting microscope. With the wires touching inside thetubing, the paperclip tip of the soldering iron is used tosmooth out the plastic and totally seal the connection (Fig.A.6).Leakage is tested for by removing the wires andattaching the needle and syringe to the 'vertical' part ofthe "T". Water is infused while observing the flow throughcatheter; therefore, the lumen size and fluid path can bedetermined. If the path is too obscure, or the lumen size103reduced too greatly, the T-piece is rejected as theprobability of clots forming in this area is much greater.Once water is within the tubing, both ends of the "T" topare clamped. A strong pressure is then applied to thesyringe while observing the connection under the microscope.The location of any leaks are noted and later sealed by re-melting the plastic (repeat steps accompanying Fig. A.6).This is continued until the leakage is eliminated. The endsare then cut leaving about 2 cm on each side of the junction(Fig. A.7). These ends are trimmed to the appropriatelength during the operation.104APPENDIX B: Unless otherwise stated, chemicals are from Sigma ChemicalCo., St. Louis, Mo., U.S.A..FORMULAS1) Modified Cortland Saline (Wolfe, 1963).Mix in order:7.25 g/L NaC10.38 g/L KC10.16 g/L CaCl20.23 g/L MgSO41.00 g/L NaHCO30.41 g/L NaH2PO41 L distilled H2O2) 2 M tris/EDTA solution2 M tris20 g/L EDTA (BDH Inc., Toronto, Ont., Canada)Combine in 1 L of HPLC grade deionized water and adjustto pH 8.6. All reagents must be HPLC grade.3) DHBA internal standard for adrenaline analysis7.704 mg DHBA (3,4 dihydroxy benzylamine hydrobromide)dissolve in 500 mL HC1 (Optima grade, FishcerScientific, Ottawa, Canada)Dilute 5000X with 0.1 N HC1. Reagents must be HPLCgrade.4) Phosphate buffered saline (PBS)1.0 g KH2PO48.5 g NaCl9.25 g Na2HPO4Dissolved in 1 L of double distilled H2O and adjustedto pH 7.45) AUC Supplement0.1 g Adenosine0.1 g Uridine0.1 g CytosineDissolved in 100 mL of Minimal Essential Medium (Gibco)and filter sterilized using a 0.45 A filter unit(Nalgene).6) G Supplement0.1g GuanosineDissolved in 100 mL Minimal Essential Medium in a warm105water bath (37 °C) and then filter sterilized.7) Tissue Culture Medium172.0 mL RPMI 1640 Medium (without L-Glutamine)20.0 mL Fetal Bovine Serum (Gibco Laboratories, NewYork)2.0 mL Non-essential Amino Acids (Whittaker,Walkersville, Ma.)2.0 mL Sodium Pyruvate (Whittaker)2.0 mL L-Glutamine (Whittaker)2.0 mL AUC Supplement2.0 mL G Supplement0.1 mL 2-mercaptoethanol 0.1 M0.2 mL Gentamicin SulphatR (Whittaker)Made aseptically in 75 cm tissue culture flask(Corning).8) Modified Barbitol Buffer (MBB)1 vial Barbital Buffer1L double distilled H2OThis produces a 5X stock. To make a working solution,add 4 parts PBS and 1 part MBB 5X stock.9) Alsever's buffer2.05 g Glucose0.8 g Tri-sodium citrate (anhydrous)0.42 g Sodium chloride100 mL double distilled waterThe solution is adjusted to pH 7.2 with 10% citric acidsolution and sterilized by filtration through a 0.22 Ammembrane.106HEMOLYTIC PLAQUE ASSAY1) Conjugation of Vibrio with Sheep RBCa) Sheep red blood cells (SRBC).Whole blood was obtained from an adult ewe from theSheep Unit, Department of Animal Science, University ofBritish Columbia and stored at a 1:1.2 ratio of blood toAlsever's buffer.b) Vibrio "0-antigen" extraction (after: Sakai et al.,1984; Kaattari and Irwin, 1985, Maule et al., 1987).The formalin killed Vibrio cells were disrupted by thefollowing method to produce an "0-antigen" for conjugationwith SRBC. Packed cells were re-suspended in a 10X volumeof PBS and boiled for 2 hours. These cells were thencentrifuged at 2000 g, at 4 °C for 30 minutes (Beckmancentrifuge). The supernatant was removed, cells re-suspended and they were both centrifuged for 45 minutes atthe previously mentioned settings. The supernatant was thendiscarded and cells re-suspended in 100 mL of 95% ETOH.This mixture was incubated for 2.5 days at 37 °C. Aftercentrifugation (10,000 g for 20 minutes), the supernatantwas discarded, and the cells washed twice with acetone. Thecells were re-suspended in a minimum amount of acetone,deposited into a plastic weigh boat and dried at 37 °Covernight.107c) Conjugation of "0-antigen" to SRBC.SRBC and "0-antigen" are combined by the followingprocedure. 8 mg of Vibrio cell wall are ground, added to 10mL PBS and boiled for 1 hour. This solution is then allowedto come to room temperature. 3 mL SRBC are placed into aculture tube and washed three times with PBS to remove theAlsever's buffer. After the final centrifugation, thesupernatant is discarded and the cells are combined with theVibrio solution. After incubation at 37 °C for 1.5 hours,the cells are centrifuged (8000 g, 4 °C for 5 minutes) andwashed in MBB four times to remove any lysed cells andunbound Vibrio. The cells are then re-suspended with MBB to10 % (ie. 0.3 mL packed cells re-suspended to a final volumeof 3 mL). The conjugated V-SRBC mixture is placed into thefridge for storage and can be successfully employed for fourdays after this procedure. Before use, the cells are washedtwo times to remove any lysed cells and then re-suspended to10% with MBB.2) Complement serumAdult coho serum was used as a source for complement.The serum was obtained in November, 1989 at the Capilanofish hatchery, North Vancouver, British Columbia. Equalaliquots of serum were stored at -80 °C until use.108LYSOZYME ASSAYa) Agarose plates0.6 mg/mL Micrococcus lysodeikticus^0.02^M NaC1^0.5^% AgaroseThe chemicals are combined in PBS (0.06 M, pH 6.0), andheated till boiling. 25 mL were then placed into plasticpetri dishes (15 cm diameter). After solidification, 9wells/plate were punched into the gel using a cork borer.The gels were allowed to dry before proceeding with theassay.b) StandardExternal standards were made using Hen egg whitelysozyme (HEWL) dissolved in PBS. One unit of lysozymeactivity was determined by a turbimetric assay to equal theamount of enzyme required to cause a 0.001 /minute decreasein A530 of a M. lysodeikticus suspension. Standards rangingfrom 100 to 4000 µg/mL were made. However, it should benoted, that as a resulting of a different optimum pH, HEWLis not considered to be a suitable standard for rainbowtrout (Mock and Peters, 1990).


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