Rainbow trout, Salmo gairdnerii Richardson, spermatozoa: effects of cations and pH on motility

Earlier work on the factors influencing inhibition of activation of the spermatozoa of Salmo gairdnerii has been confirmed. Diluents containing K⁺ and of pH less than 7.8 do not activate the spermatozoa. The presence of Na⁺ in the diluent has a mildly antagonistic effect on this inhibition. It has now been demonstrated that both Ca²⁺ and Mg²⁺ markedly antagonize the inhibition due to low pH and K^-. It is suggested that inhibition due to potassium salts could account for the inhibition previously attributed to Androgamone I.     

Purification and properties of trout gill AMP deaminase

Summary The AMP deaminase has been purified 450–500 fold from 20,000 g supernatants from trout gill. The procedure comprised cellulose phosphate and DEAE-cellulose chromatography. The gill appeared to contain different isoenzymes as indicated by different chromatographic behaviour on cellulose phosphate and different heat stabilities. The two major isoenzymes were compared with respect to their pH optima and the effect of temperature, ATP and inorganic phosphate. The pH optimum is about pH 6.7 at low substrate concentration. A second optimum is found in phosphate buffer. The substrate saturation curve is hyperbolic, even in the absence of KCl or ATP. ATP is an activator of the enzyme in the absence of KCl, but is without effect in the presence of monovalent cations. Among the monovalent cations tested, Na⁺ is the most potent activator followed by K⁺ and NH ₄ ⁺ . Inorganic phosphate is an inhibitor of gill AMP deaminase increasing the affinity for its substrate but having no effect on the maximal velocity or the Hill coefficient. The inhibition by phosphate is partially reversed by ATP. ADP and GTP are competitive inhibitors of the enzyme. In addition, the enzyme showed negative cooperativity in the presence of ATP or GTP.     

Activation of trout gill AMP deaminase by an endogenous proteinase--III. Comparative studies on the gill AMP deaminase from different fresh water and sea water teleosts

The effects of monovalent cations and inorganic phosphate, on gill AMP deaminase, were compared in different fresh water and sea water stenohaline and euryhaline Teleosts. Generally, sea water species displayed a lower sensitivity to these effectors than fresh water species. During salinity changes, the sensitivity of gill AMP deaminase to cations and phosphate were modified proportionally to the tolerance of a given species to variations of environmental salinity. In particular, these parameters were modified in the weak euryhaline, Salmo gairdneri, but not in the real euryhaline, Anguilla anguilla. In sea water adapted trout, the appearance of a modified AMP deaminase form, with similar properties to that found in sea water species, is suggested. When compared with the conclusions from the preceeding papers [Raffin (1986) Comp. Biochem. Physiol. 85B, 157-162; 85B, 163-171], the results suggest that modification of gill AMP deaminase by limited proteolysis should be a rather general adaptation mechanism to stress.     

Genetic engineering in rainbow trout, Salmo gairdnerii Richardson, by the suppression of meiotic and mitotic metaphase

Diploid gynogenesis in rainbow trout eggs was induced by a heat shock applied after fertilization with UV irradiated sperm. Survivals were high when the heat treatment was given soon after fertilization at times corresponding to the completion of egg meiosis but were low at all other times. Increases in survival occurred, however, when the heat treatment was given at 4 h 30 min and 5 h after fertilization, with an incubation temperature of 10°C, and at 9 h after fertilization, with an incubation temperature of 5°C. Electrophoretic analysis at three enzyme loci demonstrated a significant increase in homozygous offspring from eggs given these later heat treatments when compared to offspring from eggs treated between 40 and 70 min after fertilization, again with an incubation temperature of 10°C. There was still a significant frequency of heterozygotes in these groups, however, probably of spontaneous origin via diploidization during meiosis. No tetraploid alevins were observed after normal fertilization and attempted suppression of first mitosis.     

The structure of the gill of the trout,Salmo gairdneri (Richardson)

Summary A light and electron microscopic study was made of the structure of the gill arch, filament and secondary lamella of Salmo gairdneri R. Blood pathways through the gill were traced from serial histological sections, and from the examination of ink perfused tissue and perspex casts formed following resin injection of the circulatory system.The epithelium covering the gill consists of unspecialized, dark, chloride and mucous cells. The distribution of specialized cells appears to be related to gill function. The basement membrane underlying the epithelium consists of three layers, the inner collagen layer being continuous with the connective tissue core of the gills.Blood supply to the secondary lamellar respiratory surface is via branchial, filament and secondary lamellar arteries. Blood spaces of the secondary lamellae are delimited by pillar cells containing what appears to be contractile material. The marginal channel of each lamella is bounded distally by cells of endothelial origin. A network of lymph spaces within the filaments connects with efferent branchial arteries. Nutritionary capillaries within the filaments connect with afferent branchial arteries. No shunts between afferent and efferent filament arteries were found.Data from this study and previous physiological and histopathological studies suggest a mechanism for the control of blood flow to suit the respiratory requirements of the fish. This mechanism involves a system of recruitment of additional respiratory units and changes in overall blood flow patterns.This work formed part of a thesis submitted for the degree of Doctor of Philosophy in 1971 and for which M. M. was in receipt of a studentship from the Natural Environmental Research Council. The authors are grateful for the support given by research grants from the M.R.C (P.T.) and the N.E.R.C. (M.M.), and to Prof. G. M. Hughes in whose department the work was carried out.     

Changes in the fatty acid composition of steelhead trout, Salmo gairdnerii Richardson, associated with parr-smolt transformation

Fatty acids from the several lipid classes of selected steelhead trout (Salmo gairdnerii) parr and smolt tissues, previously separated by thin-layer chromatography, were analyzed by gas-liquid chromatography. The fatty acid composition of the parr was markedly different from that of the smolt; the former being characterized by relatively low amounts of polyunsaturated fatty acids and relatively high amounts of linoleic acid, much like the typical freshwater lipid pattern. The fatty acid composition of the smolt was characterized by large proportions of long-chain polyunsaturated fatty acids. Generally, the fatty acid composition of the smolt resembled the typical seawater lipid pattern. The change in fatty acid composition of the smolt is anticipatory to seawater entry and is independent of diet and water temperature. These alterations suggest that the assumption of a polyunsaturated lipid pattern during parr-smolt transformation (smoltification) is preadaptive to seawater entry.     

Comparative study on AMP deaminase in gill,muscle and blood of fish

• 1.1. High AMP deaminase activities were determined in the gill of one selachian, Scyliorhinus caniculus, and five teleosts, Anguilla anguilla, Cyprinus carpio, Salmo gairdneri, Perca fluviatilis and Esox lucius.
• 2.2. The highest activity was generally found in skeletal white muscle, except in A. anguilla and S. caniculus.
• 3.3. In s. caniculus a very high AMP deaminase activity was found in the blood where it was shown to be tightly regulated by inorganic phosphate.
• 4.4. Seasonal variations were observed for AMP deaminase activity in gill and white muscle, but also for blood Hb and protein concentration in the three tissues examined.

     

The effect of exercise on plasma cortisol and blood sugar levels in the rainbow trout, Salmo gairdnerii Richardson

Preliminary experiments were performed to determine the diurnal variation in cortisol, using trout which had been cannulated three days previously. These results indicated that cortisol levels were reasonably stable between 10.00 and 18.00 hours, thus permitting experimentation during this period without diurnal fluctuations masking the cortisol response. Uncannulated fish were exercised in a flume for 2 h at 1, 2.6 and 5 bl s^-1 and plasma samples taken from groups of five animals at 15, 30, 60 and 120 min after the start of exercise and at 1½, 12 and 24 h after the exercise ceased. The cortisol levels in all cases were elevated after 15 min, but the magnitude of the elevation increased with swimming speed. At 1 bl s^-1 the cortisol levels increased from 76.4 (± 20.4) to 129.2 (± 20.4) ng ml^-1 [mean (± s.d. )]. At 2.6 bl s^-1 the increase was from 72.4 (± 17.1) to 254.4 (± 34.4) ng ml^-1 and at 5 bl s^-1 the increase was from 69.5 (± 27.5) to 326.4 (± 39.0) ng ml^-1. The cortisol levels were stable over the exercise period and all groups recovered to baseline levels after 24 h, though the sample taken 12 h after the termination of exercise was elevated due to regular nocturnal increases in cortisol levels. There were no dramatic changes in blood sugar levels during and after exercise at 1 and 3.2 bl s^-1.     

AMP deaminase in the gill of trout (Salmo gairdneri R.). Modalities of an activation by cellular proteolytic enzymes.

1. The AMP deaminase activity present in a crude extract of trout gill increased with time. 2. Soybean trypsin inhibitor and alpha2-macroglobulin inhibited the AMP deaminase activation. NEM was also inhibitory at 10-3 M. 3. The activation process is followed by a decrease of activity which is inhibited by EGTA and enhanced by Mg2+. These two compounds were without effect on the activation process itself. 4. Trypsin induces a sharp activation of AMP deaminase in a fresh gill extract but is without effect on a fully activated extract. 5. Overall, the results suggest that neutral proteinases are implicated in AMP deaminase activation.     

Seasonal variations in plasma concentrations of 11-ketotestosterone and testosterone in male rainbow trout, Salmo gairdnerii Richardson

Measurements were made of plasma 11-ketotestosterone and testosterone in control and sex-reversed male rainbow trout, before and during their first spawning season (winter 1978/1979). Plasma concentrations of both androgens (in both groups) were 2–3 ng ml^-1 in January 1978 (apart from some precocious spawners), rose slowly to 9–11 ng ml^-1 in April and July and then increased rapidly to 100–150 ng ml^-1 in November. From this time, testosterone levels declined but those of 11-ketotestosterone continued to rise to a peak of 260 ng ml^-1 in February 1979. No significant differences in hormone levels were found between control male and masculinized female fish.     

Aspects of purine metabolism in the gill epithelium of rainbow trout, Salmo gairdneri Richardson.

1. Enzymes interconnecting the adenylate pool were present in high concentration. 2. AMP and adenosine were easily deaminated by the corresponding enzymes whose high levels were detected. 3. Adenylate was hydrolyzed either by deamination to yield IMP which was further dephosphorylated to inosine or by dephosphorylation to adenosine followed by deamination to inosine. 4. Incubation of gill extract with [-14C]-AMP in the presence and absence of ATP but with adenosine deaminase inhibitors allowed demonstration that ATP controlled the balance between these pathways. 5. Some biochemical properties of 5'-nucleotidase. AMP deaminase and adenosine deaminase were defined. 6. Purine salvage enzymes were also estimated.     

Properties of the lysosomes from liver and gill of rainbow trout,Salmo gairdnerii R.: Effect of starvation,salinity and 2, 4, 5-T

1. Three isolation procedures were used to test the labilization of the lysosomes after adaptation of the animals to seawater, starvation or acute and chronic treatments with 2, 4, 5-T, an organochlorine pesticide.2. The lysosomes from gill and liver had different properties with respect to their resistance to osmotic and mechanical shocks, or treatments with digitonin.3. Starvation induced a significant labilization of the lysosomes in liver, but not in gill.4. Salinity changes were without effect on the stability of the gill lysosomes, but induced an increase of the specific activity of the lysosomal enzyme β-acetylglucosaminidase.5. Acute treatments with 2, 4, 5-T increased lysosomal fragility in the gill but not in the liver, while chronic treatments with this herbicide increased lysosomal lability in the two tissues.6. Liver lysosomes were much more susceptible toin vitro treatments with 2, 4, 5-T than gill lysosomes.7. The results are discussed with respect to the different functions of gill and liver, their exposure to the environment, and the possible discrimination between different lysosomal populations.     

The influence of cellulose filler on feeding, growth and utilization of protein and energy in rainbow trout, Salmo gairdnerii Richardson

A nutrient source was diluted with indigestible filler in the form of α-cellulose. The trout compensated for up to 30% cellulose by increasing the total weight of food eaten, thereby stabilizing both nutrient intake and growth rate. The fish failed to compensate for levels of 40–50% cellulose and, despite evidence for an increase in the size of the stomach, nutrient intake was approximately halved. There was a slight reduction in food conversion efficiency and the growth rate declined accordingly. By contrast, faecal output rose considerably.     

Estimation of growth potential by measurement of tissue protein synthetic rates in feeding and fasting rainbow trout, Salmo gairdnerii Richardson

Protein synthesis in liver, gill and muscle tissue was measured in vivo by constant infusion of ¹⁴C-tyrosine in fed and fasted freshwater rainbow trout, Salmo gairdnerii , at 12° C. Synthesis rates (percentage of tissue protein synthesized per day) were 15-17% in liver, 4–5% in gill and 0.38% in muscle of fed fish. Liver and gill synthesis rate showed no significant change in fish that had been without food for 15 days, whereas muscle protein synthesis fell to 0.09%. The greater susceptability of muscle protein synthesis to fasting, possibly results from the greater proportion of synthesis retained as growth in this tissue. Growth rates indicate little change in protein turnover in the muscle but increased protein degradation with fasting. The difference between fed and fasted synthesis rates in muscle may be used as a measurement of potential growth rate for a particular species.     

The perchlorosoluble proteins of the serum of the rainbow trout (Salmo gairdnerii Richardson): albumin like and hemoglobin binding fraction.

1. The main perchlorosoluble fraction of rainbow trout serum has some physico-chemical characters kindred to those of human serum albumin (low molecular weight, solubility with ammonium sulfate, electrophoretic mobility, no glycoproteinic staining). 2. However, on account of obvious differences (heterogeneity and existence of various phenotypes, lack of bromophenol blue or bilirubin binding, low concentration, solubility in perchloric acid), the term "para-albumin" seems more suitable to name this compound. 3. The perchlorosoluble fraction binds hemoglobin causing an increase of peroxidasic activity. But, unlike to human haptoglobin, hemoglobin binding is partial, reversible and labile.     

Ambient ammonia effect on catalytic potential of muscle and gill AMP deaminase in fish

AMP deaminase activity was inhibited in gill and muscle of Sarotherodon mossambicus, subjected to a week-long exposure to sublethal concentration of ambient ammonia. pH-dependent kinetic studies revealed decreased catalytic efficiency of the enzyme due to altered active site density and ionization pattern in the fish tissues at different phases of ammonia exposure.