The earliest stages in the development of the circulatory system of the Rainbow Trout Oncorhynchus mykiss

The older literature suggests that the development of the blood vascular system in teleosts differs from that of other vertebrates. The evidence, however, came mostly from studies of salmonid embryos beyond the stages when blood cells had begun to circulate, which overlooked earlier developmental stages. The development of the blood vessels of the rainbow trout are illustrated from the time of the first heartbeat to the stage of eye pigment formation. Unless they can be injected with a dye solution, the earliest vessels to develop remain invisible until blood flow makes them visible. By the time of the first heartbeat stage, the embryo has a dorsal aorta, caudal artery and vein, a few transverse vessels, and even the beginning of a vitelline network. One feature peculiar to teleosts is the development of the intermediate cell mass, from which the erythrocytes and a temporary capillary network are formed rather than from the yolk sac. Development of the early posterior cardinal and the subintestinal vein occurs much as in other vertebrates. Previous investigators missed these earliest phases of development because of the difficulty of making them visible. Early formation and transformation of the vascular system of the rainbow trout generally conforms to that seen in vertebrates, except as modified by the temporary presence of the intermediate cell mass and the specialized teleostean yolk mass. With the reduction of the intermediate cell mass, the primary circulatory system for yolk utilization is transformed into a secondary one for respiratory and metabolic functions, as happens usually among vertebrates. J. Morphol. 233:215–236, 1997. © 1997 Wiley-Liss, Inc.     

A technique for repeated sampling of the blood of individual resting fish.

A dorsal cannulation technique is described. It has been employed for repeated blood sampling in unanaesthetized rainbow trout (Salmo gairdneri) kept singly in special receptacles described in the paper. The level of the studied haematological parameters (Haematocrit, Hb, glucose, lactate, K+, Na+, Ca2+) differed between fish kept in receptacles for 1 week and free-swimming fish, most probably owing to differences in the motility of the fish. The receptacle seems to minimize visual and handling disturbances, and permits both the standardization of experimental conditions and quick and easy sampling via the dorsal aorta cannula. The general variation in the blood parameter values was very small compared with the previously reported variation in such values for rainbow trout.     

Blood collection from the tail of a rat

Techniques for blood collection from the rat include puncture of the heart, retro-orbital plexus, jugular vein, saphenous vein, tail blood vessels, carotid artery, abdominal aorta, and vena cava. Most techniques (except saphenous vein and tail blood vessel puncture) require anesthesia. The following discussion focuses on two methods of blood collection - ventral tail artery puncture and dorsal or lateral tail vein puncture.     

Compliance and smooth muscle reactivity of rainbow trout (Oncorhynchus mykiss) vessels in vitro

Systemic veins have a profound influence on cardiac output in mammals. Venoregulatory mechanisms have not been adequately studied in fish and their existence has been questioned. In the present study, two characteristics of vascular mechanics, compliance and agonist-induced tension development, were investigated in rainbow trout vessels in vitro. Rapid compliance in the anterior cardinal vein and efferent branchial artery was calculated from step-wise changes in the volume-pressure curve of isolated vessel segments. Agonist-induced tension development was examined in four veins; anterior and posterior cardinals, intestinal and duct of Cuvier. Venous compliance was not altered in response to epinephrine, norepinephrine or angiotensin II, while efferent branchial artery compliance was decreased by 10^-6 mol·l^-1 epinephrine and norepinephrine but not angiotensin II. The ratios of venous to arterial compliance in vessels from two rainbow trout strains were similar (21:1 and 32:1) and consistent with the ratio reported for mammalian viens (24:1). Trout veins contracted in response to agonists in both an, agonist- and vesselspecific manner. The greatest tension per vessel wet weight was produced in anterior cardinal vein. The response pattern of anterior cardinal vein and duct of Cuvier were similar; acetylcholine, arginine vasotocin, epinephrine and norepinephrine, and the thromboxane A₂ agonist, U-44,069, produced approximately identical contractions, whereas angiotensin II was virtually ineffective. Conversely, angiotensin II was more potent than epinephrine in posterior cardinal vein. In cumulative dose-response experiments, epinephrine was equipotent in anterior cardinal vein and duct of Cuvier, whereas the latter was less sensitive to acetylcholine. Both atrial natriuretic peptide and sodium nitroprusside relaxed precontracted veins. This is the first study to determine compliance in fish vessels and the contractile nature of different rainbow trout veins. These findings suggest that venous tone and therefore cardiac output in fish may be regulated by neural or humoral mechanisms.Abbreviations ACH acetylcholine - ACV anterior cardinal vein - ANG II salmon asn¹-val⁵ angiotensin II - ANP rat atrial natriuretic peptide - AVT arginine vasotocin - DNR Department of Natural Resources - DOC duct of Cuvier - EBA efferent branchial artery - EC₅ threshold dose producing 5% maximal contraction - EC₅₀ dose producing 50% maximal contraction - EPI epinephrine - HI K⁺ 80 mmol·l^-1 - KCl IV, intestinal vein - NEPI norepinephrine - PBS phosphate buffered saline - PCV posterior cardinal vein - SNP sodium nitroprusside - U-44,069 thromboxane A₂ agonist     

Adrenergic control of venous capacitance during moderate hypoxia in the rainbow trout (Oncorhynchus mykiss): role of neural and circulating catecholamines

Central venous blood pressure (P(ven)) increases in response to hypoxia in rainbow trout (Oncorhynchus mykiss), but details on the control mechanisms of the venous vasculature during hypoxia have not been studied in fish. Basic cardiovascular variables including P(ven), dorsal aortic blood pressure, cardiac output, and heart rate were monitored in vivo during normoxia and moderate hypoxia (P(W)O(2) = approximately 9 kPa), where P(W)O(2) is water oxygen partial pressure. Venous capacitance curves for normoxia and hypoxia were constructed at 80-100, 90-110, and 100-120% of total blood volume by transiently (8 s) occluding the ventral aorta and measure P(ven) during circulatory arrest to estimate the mean circulatory filling pressure (MCFP). This allowed for estimates of hypoxia-induced changes in unstressed blood volume (USBV) and venous compliance. MCFP increased due to a decreased USBV at all blood volumes during hypoxia. These venous responses were blocked by alpha-adrenoceptor blockade with prazosin (1 mg/kg body mass). MCFP still increased during hypoxia after pretreatment with the adrenergic nerve-blocking agent bretylium (10 mg/kg body mass), but the decrease in USBV only persisted at 80-100% blood volume, whereas vascular capacitance decreased significantly at 90-110% blood volume. In all treatments, hypoxia typically reduced heart rate while cardiac output was maintained through a compensatory increase in stroke volume. Despite the markedly reduced response in venous capacitance after adrenergic blockade, P(ven) always increased in response to hypoxia. This study reveals that venous capacitance in rainbow trout is actively modulated in response to hypoxia by an alpha-adrenergic mechanism with both humoral and neural components.     

Patterns of angiogenic and hematopoietic gene expression during brown trout embryogenesis

In this article, whole mount in situ hybridization is used to examine early blood vessel and blood cell development in the embryos of the brown trout Salmo trutta lacustris. cDNAs encoding for the angiogenic markers fli1 and flk1, and for the hematopoietic markers gata1 and gata2, were identified from an expressed sequence tag library of rainbow trout. Results show that fli1, flk1 and gata2 are activated in bilateral bands of the lateral trunk mesoderm before the onset of somitogenesis, shortly followed by gata1. These bands then converge toward the ventral midline to form the intermediate cell mass (ICM) (anterior ICM). Subsequent axial vasculogenesis and initial blood cell formation involve a clear spatial separation of fli1 and gata gene expression. Fli1 staining is most intense within the axial vessel (dorsal aorta, posterior cardinal vein) forming and lateral ICM cells, whereas binding of gata1 and gata2 probes becomes confined to the central portion of ICM cells beneath the dorsal aorta. This is followed by a first wave of angiogenesis, indicated by expression of fli1 and flk1. This gives rise to the intersegmental, dorsal longitudinal anastomotic and intestinal vessels. Further angiogenesis and hematopoiesis are activated in the "posterior ICM" of the tail. Here, the absence of gata1 indicates that hematopoiesis in this tissue generates myeloid rather than erythroid cells. The results supplement and validate previous, now historical morphological work in salmonids, thus aiding the elucidation of a comprehensive general scheme of angiogenic and hematopoietic development in the teleost embryo.     

The interactive effects of a gradual temperature decrease and long-term food deprivation on cardiac and hepatic blood flows in rainbow trout (Oncorhynchus mykiss)

The aim of the present study was to determine the extent to which the fish liver is perfused with blood. Transonic? flow probes were therefore implanted around the ventral aorta and hepatic vein(s) to record baseline blood flows in rainbow trout (Oncorhynchus mykiss) previously held under two different feeding regimes (food-deprived or fed to satiation, 8-12 weeks). Fish from both groups were exposed to a gradual temperature decrease (12°C to 5°C) and physical disturbance. Cardiac output (Q), stroke volume (Sv) and hepatic venous blood flow (HVBF) were significantly reduced in food-deprived trout at 12°C. Heart rate was not significantly affected by nutritional status, but was significantly reduced when temperature was decreased to 5°C. Physically disturbing each fish at 12°C and 5°C showed that the performance capacity of the heart was not affected by food deprivation as the capacity to increase Q and Sv was not reduced in the food-deprived group. Overall this study showed that food deprivation in rainbow trout reduced cardiac and hepatic blood flows. However, long-term food deprivation did not affect the capacity of the heart to acutely increase performance.     

Effects of hypoxia on isolated vessels and perfused gills of rainbow trout.

Local hypoxia dilates systemic and constricts pulmonary blood vessels in mammals without neural or humoral involvement. The direct effects of hypoxia on isolated vessels from bony fish have not been examined. In the present study, isolated vessels (efferent branchial artery, EBA; coeliacomesenteric artery, CMA; ventral aorta, VA; and anterior cardinal vein, ACV) from rainbow or steelhead trout (Oncorhynchus mykiss) were subjected to either passive load (resting tension) or contracted with a ligand or 50 mM KCl and then subjected to 60 min of hypoxia by N(2) administration and an additional 30 min of normoxia. All vessels were usually refractory to hypoxia under conditions of resting tension. EBAs, CMAs and VAs pre-contracted with a receptor-mediated ligand were all significantly relaxed by hypoxia and only VAs recovered significantly upon subsequent restoration of normoxia. In contrast, tension in all arteries pre-contracted with 50 mM KCl was elevated further in response to hypoxia. Conversely, ligand-contracted ACVs responded to hypoxia with a further increase in tension, whereas KCl-contracted ACVs relaxed. During apparently random 2-3-week periods EBA and CMA from steelhead and EBA from rainbow trout were hyper-reactive to hypoxia. Steelhead vessels responded to hypoxia with a rapid contraction that increased in magnitude over 3 days. These contractions were independent of pre-stimulation and they were dose-dependent upon PO(2). In isolated gills, hypoxic perfusate produced an immediate but transient elevation of resistance (R(GILL)) in all four gill arches. R(GILL) increased by as much as 30% of initial values and this response was unaltered upon a second hypoxic exposure. These studies demonstrate that isolated vascular segments of rainbow trout are indeed responsive to hypoxia and that these differential responses are vessel and tone dependent and the overall response may be altered by as yet unknown seasonal or environmental factors. Hypoxia-induced arterial relaxation is blocked by elevated external [K(+)], implicating alteration of transmembrane K(+) conductance and/or membrane potential in this depressor response. K(+)-channel closure or voltage-gated Ca(2+) influx cannot account for arterial vasoconstriction due to hypoxia during KCl contractions. Vascular responses to hypoxia could have a profound impact on local flow in vivo and could mediate ventilation-perfusion matching in the branchial circulation of fish.     

Flesh qualities and muscle fiber characteristics in triploid and diploid rainbow trout

Analyzed with regard to their slaughter weights and flesh quality 78‐month‐old diploid and triploid rainbow trout (full sibs) were reared together in a pond after tagging at an age of 12 months. Triploids had higher body weights and carcass percentages than diploids (6 kg vs 4 kg and 66% vs 52%). Triploid fish also displayed lower electrical conductivity values and darker (L* value) and redder (a* value) flesh color. The fillets of the triploid trout contained more crude fat and less moisture than the diploids (6% vs 3% and 68% vs 74%, respectively). No effect of ploidy was found with regard to the protein contents. Triploid rainbow trout had larger mean white and intermediate muscle fiber areas than diploid fish in the dorsal and pelvic fin regions. In the pelvic fin part, the white muscle fiber areas were larger than in the dorsal fin part. In conclusion, adult triploid rainbow trout grow faster especially by fiber hypertrophy and have better flesh quality parameters than diploid fish.     

Angiogenic network formation in the developing vertebrate trunk

We have used time-lapse multiphoton microscopy of living Tg(fli1:EGFP)y1 zebrafish embryos to examine how a patterned, functional network of angiogenic blood vessels is generated in the early vertebrate trunk. Angiogenic vascular sprouts emerge from the longitudinal trunk axial vessels (the dorsal aorta and posterior cardinal vein) in two spatially and temporally distinct steps. Dorsal aorta-derived sprouts form an initial primary network of vascular segments, followed by emergence of vein-derived secondary vascular sprouts that interact and interconnect dynamically with the primary network to initiate vascular flow. Using transgenic silent heart mutant embryos, we show that the gross anatomical patterning of this network of vessels does not require blood circulation. However, our results suggest that circulatory flow dynamics play an important role in helping to determine the pattern of interconnections between the primary network and secondary sprouts, and thus the final arterial or venous identity of the vessels in the functional network. We discuss a model to explain our results combining genetic programming of overall vascular architecture with hemodynamic determination of circulatory flow patterns.     

The effect of environmental factors on circulation and respiration in teleost fish

Summary Anesthesia, handling and activity can produce large variations in some of the parameters of circulation and breathing movements in fish. Handling and MS222 anesthesis cause a large increase in heart rate in the tench, but have the reverse effect on the trout. Hypoxia causes a decreased heart rate and changes in dorsal aortic blood pressure. Serial sampling of the blood appears to have little effect on the parameters of circulation if the blood is replaced with saline. Removal of blood without replacement causes a decrease in blood pressure and a slowing of the heart. It is suggested that much of the variability observed in the measurement of circulatory parameters in fish can be accounted for by the experimental procedure, rather than demonstrating large inter-specific variations in teleost fish.     

Renal vasculature of the trout demonstrated by scanning electron microscopy, compared with canine glomerular vessels.

In order to gain additional information regarding renal circulatory patterns, we have used both ink and resin injections to study the arterial supply to the mesonephric kidney of trout. Arterial injections through the dorsal aorta with ink were made for histological preparations in which the length, termination and relationship of glomerular vessels were examined. Similar injections with methyl methacrylate were made in preparation of corrosion casts to provide us with gross replicas of the aortic branches to the kidney as well as casts of glomerular structure for scanning electron microscopy. The sequence of vessels through which arterial blood passed to the renal corpuscle and ultimately to the uriniferous tubules was traced. Each afferent arteriole was found to terminate in three to six branches which formed anastomosing circuits of capillaries; these vessels reunited at the hilum to form a single efferent arteriole. The efferent arterioles in trun traveled a short distance to peritubular capillary beds and sinusoids. Morphological evidence was found for preglomerular sphincter-like action only. The glomerular vessels were found to be similar to, although less complex than, those of the outer and mid-cortical regions of the dog kidney.     

Cold acclimation increases basal heart rate but decreases its thermal tolerance in rainbow trout (Oncorhynchus mykiss)

Rainbow trout (Oncorhynchus mykiss, Walbaum) were acclimated to 4 degrees C and 17 degrees C for more than 4 weeks and heart rate was determined in the absence and presence of adrenaline to see how thermal adaptation influences basal heart rate and its beta-adrenergic control in a eurythermal fish species. The basal heart rate in vitro was higher in cold-acclimated than warm-acclimated rainbow trout at temperatures below 17 degrees C. On the other hand, adaptation to cold decreased thermal tolerance of heart rate so that the maximal heart rates were achieved at 17 degrees C (75 +/- 4 bpm) and 24 degrees C (88 +/- 2 bpm) in cold-acclimated and warm-acclimated trout, respectively. Beta-adrenergic response of the heart was enhanced by cold-adaptation, since adrenaline (100 nmol l(-1)) caused stronger stimulation of heart rate in cold-acclimated (29 +/- 14%) than in warm-acclimated fish (10 +/- 1%; P = 0.03). Furthermore, adrenaline strongly opposed the temperature-dependent deterioration of force production in cold-acclimated trout but not in warm-acclimated trout. The results indicate that adaptation to cold increases basal heart rate but decreases its thermal tolerance in rainbow trout. Cold acclimation up-regulates the beta-adrenergic system, and beta-adrenoceptor activation seems to provide cardioprotection against high temperatures in the cold-adapted rainbow trout.     

Dynamic synchronization analysis of venous pressure-driven cardiac output in rainbow trout

Measurement of venous function in vivo is inherently difficult. In this study, we used the Hilbert transform to examine the dynamic relationships between venous pressure and cardiac output (CO) in rainbow trout whose blood volume was continuously increased and decreased by ramp infusion and withdrawal (I/W). The dorsal aorta and ductus Cuvier were cannulated percutaneously and connected to pressure transducers; a flow probe was placed around the ventral aorta. Whole blood from a donor was then I/W via the dorsal aortic cannula at a rate of 10% of the estimated blood volume per minute, and the duration of I/W was varied from 40, 60, 80, 90, 120, 230, 240, 260, 300, and 340 s. Compliance [change in (delta) blood vol/deltavenous pressure] was 2.8 +/- 0.2 ml x mmHg-1x g-1 (N = 25 measurements; 6 fish with closed pericardium) and 2.8 +/- 0.3 ml. mmHg-1x kg-1 (N = 19 measurements, 4 fish with open pericardium). Compliance was positively correlated with the duration of I/W, indicative of cardiovascular reflex responses at longer I/W durations. In trout with closed pericardium, CO followed venous pressure oscillations with an average time lag of 4.2 +/- 1.0 s (N = 9); heart rate (HR) was inversely correlated with CO. These studies show that CO is entrained by modulation of venous pressure, not by HR. Thus, although trout have a rigid pericardium, venous pressure (vis-a-tergo), not cardiac suction (vis-a-fronte), appears to be the primary determinant of CO. Estimation of venous compliance by ramp-modulation of venous pressure is faster and less traumatic than classical capacitance measurements and appears applicable to a variety of vertebrate species, as does the Hilbert transform, which permits analysis of signals with disparate frequencies.     

Effects of freshwater and saltwater adaptation and dietary salt on fluid compartments, blood pressure, and venous capacitance in trout

Trout are of interest in defining the relationship between fluid and salt balance on cardiovascular function because they thrive in freshwater (FW; volume loading, salt depleting), saltwater (SW; volume depleting, salt loading), and FW while fed a high-salt diet (FW-HS; volume and salt loading). The effects of chronic (>2 wk) adaptation to these three protocols on blood volume (51Cr red cell space), extracellular fluid volume (99mTc-diethylene triaminepenta-acetic acid space), arterial (dorsal aortic; P(DA)) and venous (ductus Cuvier; Pven) blood pressure, mean circulatory filling pressure (zero-flow Pven), and vascular capacitance were examined in the present study on unanesthetized rainbow trout. Blood volume, extracellular fluid volume, P(DA), Pven, and mean circulatory filling pressure progressively increased in the order SW < FW < FW-HS. Vascular capacitance in SW fish appeared to be continuous with the capacitance curve of FW fish and reflect a passive volume-dependent unloading of the venous system of FW fish. Vascular capacitance curves for FW-HS fish were displaced upward and parallel to those of FW fish, indicative of an active increase in unstressed blood volume without any change in vascular compliance. These studies are the first in any vertebrate to measure the relationship between fluid compartments and cardiovascular function during independent manipulation of volume and salt balance, and they show that volume, but not salt, balance is the primary determinant of blood pressure in trout. They also present a new paradigm with which to investigate the relative contributions of water and salt balance in cardiovascular homeostasis.     

Rainbow trout (Salmo gairdneri) stocking and Contracaecum spp

A stocking program with rainbow trout (Salmo gairdneri) at High Rock Lake, Manitoba failed due to infections with large numbers of Contracaecum spp. larvae. Nematode larvae in the intestinal tract, body cavity and musculature made the fish unmarketable. A combination of experimental infections of rainbow trout and pelicans (Pelecanus erythrorhynchos), observations on the behavior of fish-eating birds, and numbers of larval Contracaecum spp. in minnow species led to the following conclusions. The introduction of rainbow trout attracted large numbers of fish-eating birds, particularly pelicans. Concurrent predation by rainbow trout on fathead minnows (Pimephales promelas), five-spined sticklebacks (Culaea inconstans), and nine-spined sticklebacks (Pungitius pungitius), concentrated the parasites. The combined increase in densities of the introduced fish host and fish-eating birds, and the short life cycle of the parasite, increased the numbers of parasites in rainbow trout over a season and in the indigenous minnow species between years. Numbers of larvae in the indigenous minnow species declined when stocking of rainbow trout was stopped and use of the lake by fish-eating birds, particularly pelicans, returned to normal levels.     

Immunohistochemical localization of rainbow trout ladderlectin and intelectin in healthy and infected rainbow trout (Oncorhynchus mykiss)

In the present study, the pattern of immuno-reactive ladderlectin and intelectin in healthy rainbow trout is compared to rainbow trout infected with a variety of infectious agents. In healthy rainbow trout, both proteins were localized to individual epithelial cells of the gill and intestine and both proteins were clearly demonstrated within cytoplasmic granules of polymorphonuclear leucocytes and macrophages/monocytes found in blood vessels, hepatic sinusoids, renal interstitium, mucosal epithelium and submucosa of normal intestine. In tissue from infected rainbow trout, there was an overall relative increase in both lectins compared to healthy fish and both proteins were detected in extra-cellular spaces surrounding bacteria, fungi and protozoa. Increased distribution and density of both RTLL and RTInt was demonstrated along mucosal surfaces and within inflammatory leucocytes in infected tissues and immune related organs. These findings represent one of the few examples of in vivo association of defence lectins and infectious agents.     

Changes in selected blood component values of rainbow trout, Salmo gairdneri Richardson, following the blocking of the cortisol stress response with betamethasone and subsequent exposure to phenol or hypoxia

Cortisol was undetectable in rainbow trout plasma 24 h after the fish had been injected with betamethasone. This drug is known to suppress ACTH release and, consequently, cortisol production in teleosts. The blood glucose, chloride, sodium, potassium and total protein concentrations were not significantly affected by betamethasone. However, significantly increased blood packed cell volume values were found.
Betamethasone-injected fish when exposed to hypoxia or phenol had a pattern of blood component changes similar to those found in sham-injected fish also exposed to the pollutants. These changes in turn were similar to those found in exposed but uninjected and unhandled fish. Suppression of rainbow trout plasma cortisol concentrations with betamethasone apparently has no effect on the fish's short-term blood component responses to pollutants under the conditions used in these experiments, but does prevent a stressormediated increase in plasma cortisol concentrations.