Effects of osmolality and ions on the motility of stripped and testicular sperm of freshwater-and seawater-acclimated tilapia, Oreochromis mossambicus

A significantly higher concentration of testicular spermatozoa was obtained from freshwater Oreochromis mossambicus (9·9×10⁹ spermatozoa ml⁻¹) than seawater O. mossambicus (4·6×10⁹ spermatozoa ml⁻¹). The mean osmolality of the urine of freshwater fish (78·5 mOsmol kg⁻¹) was significantly different from that of seawater fish (304·8 mOsmol kg⁻¹). The mean length of the mid-piece of the spermatozoa together with the tail was more variable in freshwater O. mossambicus (8·80±0·23μm) than in seawater specimens (8·27±0·18 μm). Stripped sperm of freshwater O. mossambicus was highly contaminated by urine which was a good activator of sperm motility in O. mossambicus held in both fresh and sea water. The osmolality for initiation of motility in freshwater O. mossambicus spermatozoa was from 0 to 333 mOsmol kg⁻¹ while for seawater O. mossambicus spermatozoa it was from 0 to 1022 mOsmol kg⁻¹. The optimum osmolality for motility was from 70 to 333 mOsmol kg⁻¹ for freshwater O. mossambicus spermatozoa and from 333 to 645 mOsmol kg⁻¹ for seawater fish. In freshwater O. mossambicus spermatozoa, the presence of 20 mM CaCl₂ increased the permissive osmolality of NaCl from 184 to 645 mOsmol kg⁻¹. For seawater O. mossambicus spermatozoa, solutions of NaCl devoid of CaCl₂ were unable initiate motility, but the addition of 1·5 to 30 mM CaCl₂ to the NaCl solution (0–934 mOsmol kg₁) had a full motility initiating effect.     

Cryopreservation of the sperm of the Japanese bitterling

Sperm of the Japanese bitterling Tanakia limbata that had been cryopreserved with 5 or 10% methanol plus 95 or 90% foetal bovine serum (FBS) showed higher percentage and longer duration of motility than those that had been cryopreserved with 90% FBS and 10% DMSO, glycerol, N,N-dimethylacetamide or N, N-dimethylformamide. Foetal bovine serum, used as extender, had some cryoprotective effects when spermatozoa were cooled either with 10% methanol or without methanol. Spermatozoa, cooled to −40° C and then immersed in liquid nitrogen, had greater post-thaw motility than those cooled to −20, −60, or −80° C. The post-thaw percentage of motile spermatozoa increased significantly ( P < 0·001) with decreases in the freezing rate from 60 to 5°C min⁻¹. These results indicate that 10% methanol plus 90% foetal bovine serum is a suitable diluent for cryopreservation of bitterling spermatozoa and that samples should be cooled to -40°C at a low freezing rate for effective storage.     

Motility and fertilizing capacity of fresh and frozen-thawed spermatozoa in sturgeons Acipenser Baeri and A. ruthenus

The Siberian sturgeon ( Acipenser baeri ) and the sterlet ( A. ruthenus ) were injected with dried sturgeon pituitary (2 mg kg⁻¹), yielding 24 h later respectively 1.71 ± 0.5 and 1.65 ± 0.5 10¹¹ spermatozoa kg⁻¹ body weight. Spermatozoa were best activated with a solution of Tris HC1 50 mM, pH 8.0. The percentage of activated cells was 88 ± 4.4 in A. baeri (n = 5) and 68 ± 19 in A. ruthenus. (n = 5). In A. baeri , immediately after activation, the beat frequency of the flagellum and the mean velocity were in the range of 48–52 Hz and 100–300 μm⁻¹s, respectively. The beat frequency declined to 15 Hz at 30–40 and velocity to 100 μm s⁻¹ at 60 s post-activation. Only a small percentage of the spermatozoa remained motile after 3–4 min. In all cases spermatozoa showed mostly quasi-linear trajectories. Sperm was frozen in liquid nitrogen vapor immediately after dilution 1 v: 1 v in a cryopreservation medium (23.4 mM saccharose, 118 mM Tris-HCl pH 8.0, 20% egg yolk to which 15% DMSO were added). After fast-thawing procedure (25 s at 40°C), the percentage of motile spermatozoa (once activated in 118 mM Tris-HCl, pH 8.0) decreased to 23 ± 8.8 in A. baeri and to 15 ± 11 in A. ruthenus. The fertilizing capacity also decreased: 53 ± 8.3% vs. 89 ± 7.6% in control (P < 0.05) in A. baeri and 23 ± 11% vs 53 ± 8.3% in control (P < 0.05) in A. ruthenus. The motility pattern of the surviving frozen/thawed spermatozoa was the same as in fresh spermatozoa.     

Inhibitory effect of osmotic concentration, potassium and pH on motility of the sperm of the North American burbot Lota lota maculosa

Seminal plasma factors maintaining North American (NA) burbot Lota lota maculosa sperm quiescent were examined. Sperm were diluted into buffered saline solutions of various compositions and motility assessed. After 1 h in these solutions at 10° C, aliquots of the suspension were diluted with tap water and motility again assessed. Dilution of sperm in an incubation solution containing Ca²⁺ in the absence of K⁺ initiated sperm motility resulting in low motility when sperm were subsequently diluted in tap water. Incubation solutions with osmolalities >200 mOsm kg⁻¹ and containing 12·5 mM K⁺ prevented the onset of sperm motility and were associated with maximal sperm motility upon dilution in tap water. Sperm maintained at lower osmolalities exhibited limited motility upon dilution in tap water indicating interdependence between K⁺ and osmolality in maintaining sperm quiescent in the presence of Ca²⁺. Sperm kept in incubation solution at pH values < c. 7·5 for 1 h demonstrated reduced motility when subsequently diluted in tap water. That motility of sperm was pH sensitive was further indicated by CO₂ inhibition of motility. Therefore, NA burbot sperm are probably maintained in an immotile state, yet with potential for motility, by combination of high K⁺, osmolality and possibly pH. The results from this study differ from published information on sperm quiescence in the temporally and geographically distinct Eurasian burbot Lota lota lota .     

Seasonal differences in routine oxygen consumption rates of the bonnethead shark

Routine oxygen consumption rates of bonnethead sharks, Sphyrna tiburo , increased from 141·3±29·7 mg O₂ kg⁻¹ h⁻¹ during autumn to 218·6±64·2 mg O₂ kg⁻¹ h⁻¹ during spring, and 329·7±38·3 mg O₂ kg⁻¹ h⁻¹ during summer. The rate of routine oxygen consumption increased over the entire seasonal temperature range (20–30° C) at a Q ₁₀=2·34.     

Is tilting behaviour at low swimming speeds unique to negatively buoyant fish? Observations on steelhead trout,Oncorhynchus mykiss,and bluegill,Lepomis macrochirus

When swimming at low speeds, steelhead trout and bluegill sunfish tilted the body at an angle to the mean swimming direction. Trout swam using continuous body/caudal fin undulation, with a positive (head-up) tilt angle ( 0 , degrees) that decreased with swimming speed ( u , cm s⁻¹) according to: 0 =(164±96).u^{(−1.14±0.41)} (regression coefficients; mean±2 s.e. ). Bluegill swimming gaits were more diverse and negative (head down) tilt angles were usual. Tilt angle was −3·0 ± 0.9° in pectoral fin swimming at speeds of approximately 0.2–1.7 body length s⁻¹ (Ls⁻¹; 3–24 cm s⁻¹), −4.5 ±2.6° during pectoral fin plus body/caudal fin swimming at 1·2–1·7 L s⁻¹ (17–24cm s⁻¹), and −5.0± 1.0° during continuous body/caudal fin swimming at 1.6 and 2.5 L s⁻¹ (22 and 35cm s⁻¹). At higher speeds, bluegill used burst-and-coast swimming for which the tilt angle was 0.1±0.6°. These observations suggest that tilting is a general phenomenon of low speed swimming at which stabilizers lose their effectiveness. Tilting is interpreted as an active compensatory mechanism associated with increased drag and concomitant increased propulsor velocities to provide better stabilizing forces. Increased drag associated with trimming also explains the well-known observation that the relationship between tail-beat frequency and swimming speed does not pass through the origin. Energy dissipated because of the drag increases at low swimming speeds is presumably smaller than that which would occur with unstable swimming.     

Cardiorespiratory status of triploid brown trout during swimming at two acclimation temperatures

At 14° C, standard metabolic rate (75·1 mg O₂ h⁻¹ kg⁻¹), routine metabolic rate (108.8 mg O₂ h⁻¹ kg⁻¹), active metabolic rate ( c . 380 mg O₂ h⁻¹ kg⁻¹), critical swimming speed (U_crit 1·7 BL s⁻¹), heart rate 47 min⁻¹), dorsal aortic pressure (3·2 kPa) and ventilation frequency (63 min⁻¹) for triploid brown trout Salmo trutta were within the ranges reported for diploid brown trout and other salmonids at the same temperature. During prolonged swimming ( c . 80% U _crit), cardiac output increased by 2·3-fold due to increases in heart rate (1·8-fold) and stroke volume (1·2-fold). At 18° C, although standard and routine metabolic rates, as well as resting heart rate and ventilation frequency increased significantly, active metabolic rate and certain cardiorespiratory variables during exercise did not differ from those values for fish acclimated to 14° C. As a result, factorial metabolic scope was reduced (2·93-fold at 18° C v . 5·13-fold at 14° C). Therefore, it is concluded that cardiorespiratory performance in triploid brown trout was not unusual at 18° C, but that reduced factorial metabolic scope may be a contributing factor to the mortality observed in triploid brown trout at temperatures near 18° C.     

Cryopreservation of common carp (Cyprinus carpio L.) sperm II. Optimal conditions for fertilization

Experiments were carried out on the cryopreservation of common carp ( Cyprinus carpio L.) sperm. Optimal conditions for fertilization including suitable medium and sperm:egg ratio were determined. Sperm was diluted in modified Kurokura's 'Extender 2'containing DMSO as cyroprotectant in 10% final concentration. The dilution rate was 1:9 (sperm:diluent). Sperm was diluted and equilibrated (10 min) at 2°C. Sperm was then frozen in plastic straws (0.5 ml) at the following rate: 0°C–4°C: 4°C min⁻¹; −4°C–80°C: 11°C min⁻¹; from −80°C they were plunged directly into liquid nitrogen (− 196°C). Frozen samples were thawed in a water bath at 40°C. Fertilization rates achieved were much higher in water than in other solutions. Optimal ratios of frozen sperm:egg:water (1:20:20 in volume) and optimal number of frozen spermatozoa:egg (10⁵ spz: 1 egg) were determined. In such conditions, a strong positive correlation (c =+0.846) was found between the post-thaw motility and the fertility of frozen sperm securing high fertilization (99.6%, percent of control). No significant difference was found between fertilization and hatching rates achieved using frozen-thawed common carp sperm.     

The angle of attack of the body of common bream while swimming at different speeds in a flume tank

The body attack angle of common bream Abramis brama varied with swimming speed and was best described by θ = −3·32 (±0·24) − 9·23 (±0·54)e^{− u} ( r ² = 0·56, P  < 0·0001; ±1 s . e . given in parentheses), where θ is the body attack angle and u is swimming speed. The hypothesis that neutrally buoyant fishes may swim with body attack angles deviating increasingly from 0° as the swimming speed decreases is supported.     

Stimulation of biomethanation by Clostridium sp. PXYL1 in coculture with a Methanosarcina strain PMET1 at psychrophilic temperatures

Aim:  Bioaugumentation of low temperature biogas production was attempted by addition of cold-adapted Clostridium and a methanogen.
Methods and Results:  A psychrotrophic xylanolytic acetogenic strain Clostridium sp. PXYL1 growing optimally at 20°C and pH 5·3 and a Methanosarcina strain, PMET1, growing optimally on acetate and producing methane at 15°C were isolated from a cattle manure digester. Anaerobic conversion of xylose at 15°C with the coculture of the two strains was performed, and batch culture methane production characteristics indicated that methanogenesis occurred via acetate through 'acetoclastic' pathway. Stimulation studies were also undertaken to evaluate the effect of exogenous addition of the coculture on biogas yields at 15°C. Addition of 3 ml of PXYL1 at the rate of 12 × 10² CFU ml⁻¹ increased the biogas 1·7-fold (33 l per kg cowdung) when compared to control (19·3 l per kg cowdung) as well as increased the volatile fatty acid (VFA) levels to 3210 mg l⁻¹ when compared to 1140 mg l⁻¹ in controls. Exogenous of addition of 10 ml PMET1 inoculum at the rate of 6·8 ± 10² CFU ml⁻¹ in addition to PXYL1 served to further improve the biogas yields to 46 l kg⁻¹ as well as significantly brought down the VFA levels to 1350 mg l⁻¹.
Conclusions:  Our results suggest that the rate-limiting methanogenic step at low temperatures could be overcome and that biogas yields improved by manipulating the population of the acetoclastic methanogens.
Significance and Impact of the Study:  Stimulation of biomethanation at low temperature by coculture.     

Growth, diet and metabolism of common wolf–fish in the North Sea, a fast–growing population

The von Bertalanffy growth parameters for common wolf–fish Anarhichas lupus in the North Sea were: male: L ∞=111·2 cm, t ₀=–0·43 and K =0·12; and female: L ∞=115·1 cm, t ₀=–0·39 and K =0·11, making this the fastest growing stock reported. Resting metabolic rates (RMR±S.E.) and maximum metabolic rates (MMR±S.E.) for six adult common wolf–fish (mean weight, 1·39 kg) at 5° C were 12·18±1·6 mg O₂ kg^–1 h^–1 and 70·65±7·63 mg O₂ kg^–1 h^–1 respectively, and at 10° C were 25·43±1·31 mg O₂ kg^–1 h^–1 and 113·84±16·26 mg O₂ kg^–1 h^–1. Absolute metabolic scope was 53% greater at 10° C than at 5° C. The diet was dominated by Decapoda (39% overall by relative occurrence), Bivalvia (20%) and Gastropoda (12%). Sea urchins, typically of low energy value, occupied only 7% of the diet. The fast growth probably resulted from summer temperatures approximating to the optimum for food processing and growth, but may have been influenced by diet, and reduced competition following high fishing intensity.     

The effect of temperature fluctuations on oxygen consumption and ammonia excretion of underyearling Lake Inari Arctic charr

Underyearling Lake Inari Arctic charr Salvelinus alpinus were acclimated to 11·0) C for 3 weeks, and then one group was maintained at 11·0) C and others were exposed to 14·4) Cconst, 17·7) C_const or a diel fluctuating temperature of 14·3° C ± 1° C (14·3° C_fluc). Routine rates of oxygen consumption and ammonia excretion were measured over 10 days before the temperature change and over 31 days following the change. Measurements were made on fish that were feeding and growing. The temperature increase produced an immediate increase in oxygen consumption. There was then a decline over the next few days, suggesting that thermal acclimation was rapid. For groups exposed to constant temperature there was an increase in oxygen consumption ( M _accl, mg kg⁻¹ h⁻¹) with increasing temperature ( T ), the relationship being approximated by an exponential model: M _accl= 46·53e0·^{086 T} . At 14·3° C_fluc oxygen consumption declined during the 3–4 days following the temperature shift, but remained higher than at 14·4° C_const. This indicates that small temperature fluctuations have some additional influences that increase metabolic rate. Ammonia excretion rates showed diel variations. Excretion was lower at 11° C_const than at other temperatures, and increases in temperature had a significant effect on ammonia excretion rate. Fluctuating (14·3° C_fluc) temperature did not influence ammonia excretion relative to constant temperature (14·4° C_const).     

Motility of spermatozoa from shovelnose sturgeon and paddlefish

The spermatozoa in the seminal plasma from shovelnose sturgeon Scaphirhynchus platorynchus and paddlefish Polyodon spathula were immotile with only a few spontaneously motile spermatozoa for 5-10 and 10-20 s, respectively. Spermatozoa of shovelnose sturgeon were observed to be 100% motile immediately after sperm dilution in 10 m m NaCl and 20 m m Tris-HCl, pH 8.5. The duration of mass progressive movement was 2-3 min; and 1 to 5% of spermatozoa remain active after 360 s (P<0.01). Spermatozoa of paddlefish demonstrated the best motility 10 s after dilution in 10 m m NaCl with 20 m m Tris-HCl, pH 8.5. The duration of mass progressive movement was 2-3 min and 1 to 5% of spermatozoa remained active after 370 s ( p <0.01). The spermatozoa of shovelnose sturgeon and paddlefish were motile in a range of osmotic pressure from 0 to 100 mosmol kg⁻¹ and 0 to 120 mosmol kg⁻¹, respectively. The best results with short-term storage of sperm from shovelnose sturgeon and paddlefish were observed in 100 m m glucose + 20 m m Tris-HCl, pH 8.5 and 150 m m glucose + 20 m m Tris-HCl, pH 8.5.     

Salinity adaptation in the snakehead, Ophiocephalus maculatus Lacépède: changes in oxygen consumption, branchial Na+-K+-ATPase and body composition

Snakeheads were adapted to fresh water (1 mOsm kg⁻¹), 25% sea water (230 mOsm kg⁻¹), 33% sea water (320 mOsm kg⁻¹) and 40% sea water (380 mOsm kg⁻¹) for 20 days. Exposure to salt water resulted in tissue dehydration, elevations of plasma osmolality, Na⁺, Mg²⁺, Cl⁻and protein concentrations and stimulation of branchial Na⁺-K⁺-ATPase activity. These changes were accompanied by concomitant decline of the hepatosomatic index and liver glycogen concentration. The routine rate of oxygen consumption was increased in snake-heads adapted to 33% sea water. These data were taken to indicate a stressful effect of salinity to the snakehead despite documentation of its ability to penetrate into brackish waters.     

Effects of social and visual contact on the oxygen consumption of juvenile sea bass measured by computerized intermittent respirometry

The resting metabolic rate (RMR) of juvenile European sea bass Dicentrarchus labrax L. (47·5±1·5 g, 15–18 cm) was 126·2±2·5 mgO₂ kg⁻¹ h⁻¹, and temporal patterns of oxygen consumption were not affcted by visual contact or social interaction with conspecifics. The results suggest that a group effect is not present in juvenile D. labrax , thus no selective advantage of shoaling is gained through lowered metabolism in this facultative schooling species.     

Sperm structure and motility of the freshwater teleost Cottus gobio

When motility of spermatozoa of Cottos gobio was initiated with distilled water, the motility rate decreased to 0% within 1 min, and significant signs of osmotic alterations were observed at the end of the motility period. By contrast, in 50 mmol 1⁻¹ NaCl solution, the motility rate persisted for 120–140 min. In both distilled water and in 50 mmol 1⁻¹ NaCl solution, the main swimming type of spermatozoa was linear motion during the whole motility period. The initial swimming velocity (50.0 ± 2.1 μm s⁻¹) measured 10 s after motility initiation was similar in both distilled water and in 50 mmol 1⁻¹ NaCl solution. In distilled water, the velocity decreased to <20 μm s⁻¹ (locally motile) during the first minute of the motility phase. In 50 mmol 1⁻¹ NaCl solutions, it remained at a constant level during the first 60 min of the motility period, but then started to decrease to <20 μm s⁻¹ after 120 min. When 5 mmol 1⁻¹ potassium cyanide, antimycin or atractyloside was added to the 50 mmol 1⁻¹ NaCl solution, the motility period was reduced to ≤2min. Ten millimoles per litre 2-deoxy-D-glucose, malonate or a mixture of 5 mmol 1⁻¹ atractyloside and 5 mmol 1⁻¹ carnithine did not effect the duration of the motility period. This indicates that sperm energy metabolism depends mainly on respiration rate and fatty acid metabolism.     

Mathematical modelling of the heat resistance of Listeria monocytogenes

The heat resistance of Listeria monocytogenes phagovar 2389/2425/3274/2671/47/108/340 (1992 French outbreak strain) in broth was studied at 55, 60 and 65 °C. Experiments were carried out on bacterial cultures in three different physiological states: cultures at the end of the log phase, cultures heat-shocked at 42 °C for 1 h, and subcultures of cells resistant to prolonged heating. Survivor curves were better fitted using a sigmoidal equation than the classical log-linear model. This approach was justified by the existence of heat resistance distributions within the bacterial populations. Peaks (log₁₀ of heating time) of heat resistance distributions of untreated, heat-shocked, and selected cultures at 55, 60 and 65 °C were 0·34, −0·90 and −1·84 min, 0·74, −0·51 and −1·24 min, and 0·17, −0·94 and−1·45 min, respectively. The widths of the distributions are proportional to 0·29, 0·36and 0·41 min^0·5, 0·26, 0·36 and 0·41 min^0·5, and 0·34, 0·44 and 0·41 min^0·5. An increase in thethermal tolerance could then be induced by sublethal heat shock or by selection of heatresistant cells.     

Assessment of microbial involvement in the elevation of copper levels in drinking water

A study of bacterial populations in metropolitan Adelaide domestic reticulation pipes was conducted to investigate a possible link between copper in drinking water and biofilms. Biofilm densities from cold water copper pipes at 10 sample sites were measured by viable cell counts. The range detected was from <2 × 10¹ to 3·25 × 10⁷ cfu cm⁻². Five isolates were selected for further experiments as they represented a range of responses to solvated copper and relative tendency for adhesion on glass slides. Drinking water supplied to the Adelaide Hills is high in total organic carbon (TOC; 22·57 mg C l⁻¹) and has a negative Langelier Index (LI; −1·16), whereas Adelaide metropolitan water undergoes filtration and has both a lower TOC and LI (10·72 mg C l⁻¹, LI, −0·49). Copper coupons were exposed to biofilm isolates (24 h), washed and resuspended in Adelaide metropolitan and Adelaide Hills water. Copper coupons not exposed to biofilm isolates were suspended in respective waters as a control. After 5 d of incubation, the copper content of Adelaide Hills water (4·71 ± 0·87 mg Cu l⁻¹), in which the copper coupons were suspended, consistently exceeded values obtained in the metropolitan Adelaide water (1·17 ± 0·249 mg Cu l⁻¹). The concentration of copper in the Adelaide Hills water was influenced by the bacterial species forming the biofilm on the coupon, with Agrobacterium sp. producing significantly higher levels of soluble copper than the control. The experiments reported here indicate that the suspended organic carbon, the aggressivity of the water and the biofilm may independently or synergistically increase the dissolution of copper from pipes into drinking water.     

Formation of the enveloping layer of the chum salmon egg in isotonic salt solutions

After stimulation in a hypotonic solution (9.4 mOsm kg⁻¹), inseminated eggs of the chum salmon Oncorhynchus keta initiate cleavages in isotonic salmon Ringer's solution (267.3 mOsm kg⁻¹) containing 3.2 mM Ca²⁺ ions. Blastomeres of these eggs, however, separate from each other and the enveloping layer is not observed at the blastula stage. An increase in external divalent cations rescues the separation; the concentration of CaCl₂ in the external medium should be 25 mM or more to induce close contact of blastomeres and the formation of an enveloping layer in isotonic salt solutions. The effectiveness of Ca²⁺ ions can be substituted by Mg²⁺, Sr²⁺ and Zn²⁺ ions; the same results are obtained in isotonic MgCl₂ and SrCl₂ solutions (100 mM) or in isotonic salmon Ringer's solution containing Zn ions (6.2 mM). The close contact of blastomeres and the formation of an enveloping layer are also observed in a low Ca²⁺ concentration (< 0.1 mM) in a hypotonic salt solution (9.4 mOsm kg⁻¹). The Ca²⁺ level in the external medium to induce the enveloping layer formation seems to be correlated with the salinity of the incubation medium. It is suggested that adhesion molecules on the surface of blastomeres in the chum salmon eggs are different in properties from those found in sea urchin and other fish species.     

Cold hardiness in the black rice bug, Scotinophara lurida

Abstract.  The mechanisms and strategies for winter survival of the black rice bug Scotinophara lurida are investigated along with the relationship between cold hardiness and diapause. The ability of S. lurida to survive subzero temperatures varies depending on developmental stage, temperature and exposure duration. Mean supercooling point (SCP) varies from –7.6 to –10.7 °C with developmental stage, but is not significantly different between stages examined. The SCP also varies with season, being lowest in January and increasing rapidly in February and remaining almost at the same level (–7.3 to 9.6 °C) until April The osmolality of haemolymph of field-collected S. lurida adults rises dramatically from 53.9 mOsm kg⁻¹ in August to 75.3 mOsm kg⁻¹ in December, and then declines linearly to 57.0 mOsm kg⁻¹ in May. Field-collected S. lurida adults show a peak glucose content in October, glycerol content in November and trehalose content in December. Only trehalose content decreases after the application of the juvenile hormone analogue, fenoxycarb, suggesting that trehalose is a cryoprotectant during diapause. These various physiological and biochemical traits related to cold tolerance in S. lurida may be, at least in part, under the control of juvenile hormone through the reproductive diapause programme.