Cryopreservation of common carp (Cyprinus carpio L.) sperm I. The importance of oxygen supply

Experiments were carried out on the cryopreservation of common carp ( Cyprinus carpio L.) sperm. The effects of pre-freezing oxygen supply on post-thaw motility and the efficacy of different extenders were studied. Sperm diluents contained DMSO as a cryoprotectant in 10% final concentration. The dilution rate was 1:9 (sperm:diluent). Sperm was diluted and equilibrated (10 min) at 0°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, straws were plunged directly into liquid nitrogen (−196°C) for further storage. Frozen samples were thawed in a water bath at 40°C.
The freezability of common carp sperm showing reduced motility (due to suboptimal oxygen supply) after transportation could be restored when 30 min of oxygenation was applied prior to freezing. Highest post-thaw motility (57%, percent of control) was achieved when sperm was diluted with modified Kurokura's 'Extender 2'.     

Motility and fertilizing capability of cryopreserved Acipenser ruthenus L. sperm

Motility before and after cryopreservation of Acipenser ruthenus sperm was determined by computer-assisted sperm motion analysis (CASA). In addition, fertility of fresh and frozen/ thawed sperm was tested by in vitro fertilization. Sperm of A. ruthenus was successfully cryopreserved by 1/1 (v/v) dilution with ethylene glycol in final concentrations from 12.5% to 20%. Stepwise cooling using a programmable freezer and fast thawing in a water bath (40°C) for 3 s and 5 s, respectively, resulted in motility rates of 10% to 28% and fry yields of 44% to 94% compared to controls (fresh sperm). With fresh material, different sperm to egg ratios varying from 10³: 1 to 10⁶: 1 gave similar fertilization rates of more than 90%. Using this cryopreservation method the establishment of sperm banks for endangered sturgeon species and the development of a fertility test for seasonally independent testing seem to be possible.     

Field ecology of freeze tolerance: interannual variation in cooling rates, freeze-thaw and thermal stress in the microhabitat of the alpine cockroach Celatoblatta quinquemaculata

Microclimate recordings were made over four years on the Rock and Pillar Range, New Zealand. These are used in conjunction with mortality and freezing data derived in the laboratory to make inferences about the winter thermal ecology of the freeze-tolerant alpine cockroach Celatoblatta quinquemaculata (Dictyoptera: Blattidae). Threshold temperatures are identified through laboratory experiments at ecologically relevant cooling rates: 0°C; −3.1°C (FP₅: the temperature at which 5% of cockroaches are expected to be frozen); and −4.5°C (FP₅₀). The maximum cooling rate in the microhabitat across any of the thresholds was 0.06°C min⁻¹, considerably slower than the 1°C min⁻¹ normally used in laboratory studies. Freeze-thaw events occurred regularly in the field, and temperature minima occasionally fell to temperatures lethal to C. quinquemaculata . Variability in snow cover contributed to interannual variation in microclimate temperatures. Decreased snow cover is predicted with climate change scenarios, and this will probably result in more freeze-thaw cycles and more extreme minimum temperatures in this environment. It is concluded that the limited environmental tolerances of the animals living in this habitat make the few degrees of interannual variation ecologically relevant.     

Sperm motility and cryopreservation of spermatozoa in freshwater gobies

Testicular sperm motility and methods for the cryopreservation of spermatozoa in the freshwater goby Rhinogobius sp. CB (Cross Band type) were examined. Spermatozoa were almost immotile upon dilution with 300 mOsm kg⁻¹ of NaCl, KCl and mannitol solutions but began to swim in solutions with concentrations <200 mOsm kg⁻¹. The highest percentage and longest duration of motility was obtained in the 0 and 100–200 mOsm kg⁻¹ solutions, respectively. The highest post-thaw motility, c. 50% of motility before cryopreservation, was obtained when spermatozoa were diluted with an extender of 10% methanol and 90% artificial seminal plasma, cooled at −10·0 ± 1·1° C min⁻¹ (mean ± s . e .) to −50° C and plunged into liquid nitrogen. Spermatozoa were cryopreserved in a 50 μl acrylic haematocrit tube to store the small amount of milt. As the cryopreservation method described above was applicable to the endangered Rhinogobius sp. BI (Bonin Island type), it is probable that this method can be used for other species of freshwater gobies.     

Macromolecular synthesis by yeasts under frozen conditions

Although viable fungi have been recovered from a wide variety of icy environments, their metabolic capabilities under frozen conditions are still largely unknown. We investigated basidiomycetous yeasts isolated from an Antarctic ice core and showed that after freezing at a relatively slow rate (0.8°C min⁻¹), the cells are excluded into veins of liquid at the triple junctions of ice crystals. These strains were capable of reproductive growth at −5°C under liquid conditions. Under frozen conditions, metabolic activity was assessed by measuring rates of [³H]leucine incorporation into the acid-insoluble macromolecular fraction, which decreased exponentially at temperatures between 15°C and −15°C and was inhibited by the protein synthesis inhibitor cycloheximide. Experiments at −5°C under frozen and liquid conditions revealed 2–3 orders of magnitude lower rates of endogenous metabolism in ice, likely due to the high salinity in the liquid fraction of the ice (equivalent of ≈ 1.4 mol l⁻¹ of NaCl at −5°C). The mesophile Saccharomyces cerevisae also incorporated [³H]leucine at −5°C and −15°C, indicating that this activity is not exclusive to cold-adapted microorganisms. The ability of yeast cells to incorporate amino acid substrates into macromolecules and remain metabolically active under these conditions has implications for understanding the survival of Eukarya in icy environments.     

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 .     

Measurement of the kinetics of bacterial adherence to hexadecane in polystyrene cuvettes

Abstract The kinetics of bacterial adherence to hexadecane were measured using disposable polystyrene cuvettes. The rate of adherence was exponential, and was itself linearly dependent on the water:hexadecane ratio employed. The dependency of the rate of adherence on the water:hexadecane ratio, termed the removal coefficient, varied from 4.7 min⁻¹ for Streptococcus pyogenes to 72 min⁻¹ for Acinetobacter calcoaceticus . The removal coefficient of Serratia marcescens was a function of growth temperature, 48 min⁻¹ following growth at 30°C as opposed to 5.8 min⁻¹ for 35°C-grown cells.     

The minimum effective spermatozoa : egg ratio for artificial insemination and the effects of mercury on sperm motility and fertilization ability in Clarias gariepinus

The optimal ratio of spermatozoa : egg (15 000 : 1) for artificial insemination of African catfish Clarias gariepinus gave fertilization and hatching rates of 80 and 67%, respectively. Below a sperm : ova of 3000 : 1 fertilization success decreased significantly. Excessive sperm (>15 000 : 1) partly inhibited fertilization success. Sperm motility was decreased significantly by 0·001 mg 1⁻¹ Hg²⁺ as HgCl₂, but its effect on fertilization was dependent on the sperm : ova ratio, since excess sperm masked the effect of the pollutant. The most sensitive sperm : ova ratio for monitoring pollutant effects on fertilization success was 1500 : 1, which corresponds to half the minimal amount that yields a high fertilization rate in artificial insemination. There was a good correlation between fertilization and hatching rates ( r =0·83; P<0·05). Although both fertilization and hatching rates provide equally good indicators of fertilization success, the more rapid fertilization rate test is recommended since it requires only 12 h.     

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.     

Ultrastructural changes arising from freezing of leaf blade cells of rye (Secale cereale): an investigation using freeze-substitution

The survival at sub-zero temperatures of leaf blade cells of rye ( Secale cereale L. cv. Voima), which had not been cold acclimated, was determined by measuring the efflux of ninhydrin-positive substances: 50% of the cells were dead at −4°C (LT₅₀) and none survived at −12°C or below. Examination of ultrastructural changes during cold hardening and freezing injury requires frozen tissues prepared for transmission electron microscopy without thawing. Specimens were prepared from leaf blade segments at room temperature, −4°C or −12°C by plunge freezing at 3 m s⁻¹ into a cooling medium at −170°C followed by freeze-substitution in acetone with OsO₄ fixation. Comparisons of room temperature specimens were made with those prepared by chemical fixation using glutaraldehyde/paraformaldehyde/tannic acid. On freezing to −12°C, the cells were severely dehydrated and distorted, the vacuoles severely shrunken and the cytoplasm and mitochondria disorganized whereas the chloroplasts were little affected. On freezing to −4°C, some cells were as disorganized as those at −12°C, others were relatively intact, and some showed evidence of intracellular ice crystal formation.     

Hypometabolism in torpid goldsinny wrasse subjected to rapid reductions in seawater temperature

Goldsinny Ctenolabrus rupestris were subjected to rapid, environmentally realistic, reductions in temperature at 2° C increments from 10 to 4° C over a 3-day period in full-strength sea water. In separate experiments, oxygen uptake measurements and ultrasound recordings of heart rate and opercular motion were carried out at regular intervals over the same temperature regime. Mean oxygen uptake rates fell from 0.042 to 0.028 ml O₂ g⁻¹ h⁻¹ between 10 and 6° C respectively (Q₁₀=2.71). Between 6 and 4° C mean rates decreased from 0.028 to 0.008 ml O₂ g⁻¹ h⁻¹ (Q₁₀=542). Mean opercular motion and heart beat rates decreased from 49.5 and 60.3 beats min⁻¹ respectively at 10° C to 18.7 and 18.0 beats min⁻¹ respectively at 4° C. Most goldsinny subjected to 4° C were observed in a torpid state and would not react to external stimulation. Opercular motion was erratic at 4° C and would at times cease altogether for periods up to 1.3 min duration. Heart movement was diffcult to detect at 4° C and may also have ceased for prolonged periods. Q₁₀ values for opercular motion and heart beat rates recorded between 6 and 4° C were 6.39 and 24.52 respectively compared with values of 2.42 and 2.93 respectively recorded between 10 and 8° C. Such large depressions in metabolism appear not to have been reported previously for a marine fish species. No goldsinny mortalities were recorded at any temperature. The possibility that hypometabolic torpor is an adaptive strategy for goldsinny survival at low environmental temperatures is discussed.     

Cryopreservation of leucocytes of channel catfish for subsequent cytogenetic analysis

Channel catfish leucocytes cryopreserved with glycerol or dimethyl sulphoxide (DMSO) had significantly higher ( P <0.05) viability and recovery rates than did cells cryopreserved with methanol. After 7 days of frozen storage, a 24 to 27% reduction of viability was observed for cells cryopreserved with glycerol; a 25 to 43% reduction for cells frozen with DMSO, and a 67 to 100% reduction for cells frozen with methanol. The concentration of cryoprotectants affected the viability of cryopreserved cells significantly ( p <0.05). The viability reduction was 36% for cells frozen with 5% of cryoprotectants, 30% for cells frozen with 10% of cryoprotectants, and 49% for cells frozen with 15% of cryoprotectants. The viability of cells frozen at the slower rate (-2.7°C min⁻¹) was significantly higher ( p <0.05) than that of cells frozen at the faster rate (-45°C min⁻¹). Best results were obtained for cells cryopreserved with 10% of glycerol or DMSO and frozen at the slower rate. The chromosomes prepared from cells cryopreserved using this procedure were identical to those prepared from fresh cells, and to those reported in the literature for channel Catfish.     

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.     

The effect of temperature and mass on routine metabolism in Sarotherodon (Tilapia)mossambicus (Peters)

A reappraisal of oxygen uptake by Sarotherodon mossambicus was undertaken using a continuous flow respirometer. Measurements were obtained over the temperature range 16°C–37°C for fish weighing between 10 g and 150 g. Oxygen uptake was converted to energy equivalents ( Q _ox) using the value 13.68 J mg O₂^–1and the routine metabolic energy expenditure can be described by the equation E =0.0086 t ^{2 0783} M ^{0 652} where E is the energy requirement for routine metabolism expressed in J h^-1, t the temperature in °C and M the mass in g.     

THE COMBINED EFFECTS OF LOW TEMPERATURE AND SO2+NO2 POLLUTION ON THE NEW SEASON'S GROWTH AND WATER RELATIONS OF PICEA SITCHENSIS

Picea sitchensis (Bong.) Carr. seedlings were exposed to SO₂, NO₂ and SO₂+ NO₂ during dormancy in controlled environments, and were taken to night temperatures of 4, 0, −5, −10 and −15 °C in a freezer. Conditions in the freezer were carefully monitored during the low–temperature treatments. In two experiments, different photoenvironments and temperature regimes were imposed prior to the cold treatments, and different effects were observed. In the first, only limited frost hardiness was achieved and night temperatures of −15 °C were lethal. Temperatures of −5 and − 10 °C led to poor survival of lateral buds, particularly in plants exposed to 45 ppb SO₂. The poor bud break in plants exposed to SO₂ and to − 5 °C resulted in a loss of the effectiveness of this temperature as a chill requirement. Pressure-volume analysis showed that the shoots of plants exposed to NO₂ had greater elasticity (lower elastic moduli, e), so that loss of turgor occurred at lower relative water contents. In contrast, a hardening period (2 weeks in night/day temperatures of 3/10 °C and 8 h days at 50 μmol m⁻² s⁻¹ PAR) gave decreased elasticity and lower solute potentials of spruce shoots. In the second experiment, exposure to 30 ppb SO₂ and SO₂+ NO₂ led to slight, but consistent, increases in frost injury to the needles of plants frozen to − 5 and − 10 °C. The results suggest that the main interaction of low temperatures and winter pollutants may be on bud survival rather than on needle damage, but that effects are subtle, only occurring with certain combinations of pollutant dose and cold treatment.     

Physiological adaptation to aridity in the bushveld gerbil, Tatera leucogaster

Oxygen consumption, rectal temperature, thermal conductance, and evaporative water loss (EWL) were determined in resting captive Tatera leucogaster at ambient temperatures of between 14 and 38 °C. Basal metabolic rate (BMR) was 0.86 ml O₂. min⁻¹ (S.D.=0.15, n = 6), 45% of that expected for a rodent of the same body mass (106.2 g). Minimum wet thermal conductance was 0.21 ml O₂. min⁻¹, °C⁻¹ (S.D. = 0.01, n = 6), 113% of that expected for a mammal of the same body mass. Wet thermal conductance increased exponentially at temperatures greater than 32 °C. Mean rectal temperature was 35.3 °C below 35 °C (S.D. = 0.5, n = 6) and 39.3 (S.D. = 0.6, n = 5) at 38 °C. Mean resting EWL was 1.43 mg. min⁻¹ (S.D. = 0.14, n = 6) between 15 and 32 °C and increased dramatically at temperatures above 32 °C. Combining our data with data from the literature suggests that gerbils (Family Muridae; subfamily Gerbillinae) have, on average, low BMR and average minimum wet thermal conductance when compared to other rodents and other mammals, respectively, of the same body mass. Similarly, rodents (including gerbils) from arid habitats have, on average, lower rates of EWL when at rest below thermoneutrality than do other rodents of the same body mass from mesic habitats.     

Characteristics of sperm of captive seabass in relation to its fertilization potential

Seabass Dicentrarchus labrax sperm concentration was high (up to 60 × 10⁹ spz ml⁻¹) but decreased significantly at the end of the reproductive season (mid-March) in monthly sampled fish. The spermiation period may be shortened by frequent stripping. Sperm can be prediluted up to 1: 128 in non-activating medium without loss of initial motility and motility duration. Immediately after activation by transfer to sea water, all the spermatozoa were motile for 10 s and then the number of motile cells decreased progressively but sharply to zero, so that the duration of sperm motility was very short (40 s). As a consequence, the fertility of seabass sperm decreased exponentially after 10 s following sperm activation and was zero by 1 min. The sperm requirements for optimal fertilization were c . 66 000 spermatozoa per egg. Scalingup of the experimental insemination procedure yielded better fertilization rates while conserving the individual differences due to the breeder pairs.     

Effect of long-term and rapid cold hardening on the cold torpor temperature of an aphid

Abstract.  The effect of long-term (seasonal) acclimation and rapid cold hardening is investigated on the cold torpor temperature ( CT _min) of adult grain aphids, Sitobion avenae, reared at 20 or 10 °C for more than 6 months before experimentation. Rapid cold hardening is induced by exposing aphids reared at 20 to 0 °C for 3 h and aphids reared at 10 to 0 °C for 30 min (acclimation regimes previously found to induce maximum rapid cold hardening). The effect of cooling aphids from the same rearing regimes from 10 to −10 °C at 1, 0.5 and 0.1 °C min⁻¹ is also investigated. In the 20 °C acclimated population, rapid cold hardening and cooling at 0.1 °C min⁻¹ both produce a significant decrease in CT _min from 1.5 ± 0.3 to –0.9 ± 0.3 and –1.3 ± 0.3 °C, respectively. Rapid cold hardening also results in a significant reduction in CT _min of the population reared at 10 °C from 0.8 ± 0.1 to –0.9 ± 0.2 °C. However, none of the cooling regimes tested reduces the CT _min of the winter-acclimated (10 °C) population. The present study demonstrates that rapid cold-hardening induced during the cooling phase of natural diurnal temperature cycles could lower the movement threshold of S. avenae , allowing insects to move and continue feeding at lower temperatures than would otherwise be possible.     

Effect of temperature on swimming performance of sea bass juveniles

At four temperatures ( T= 15, 20, 25 and 28° C) swimming performance of Dicentrarchus labrax was significantly correlated with total length (23–43 mm L _T); r²=0.623–0.829). The relative critical swimming speed ( RU _crit= U _crit L _T⁻¹), where U _crit is the critical swimming speed, was constant throughout the L _T range studied. The significant effect of temperature on the relative critical swimming speed was described binomially: RU _crit=−0.0323T²+ 1.578 T −10.588 (r²=1). The estimated maximum RU _crit (8.69 L _T s⁻¹) was achieved at 24.4° C, and the 90% performance level was estimated between 19.3 and 29.6° C.     

PATTERNS OF INTERSPECIFIC INTERACTIONS IN THE ULVA-DOMINATED INTERTIDAL COMMUNITY IN A SOUTHERN COAST OF KOREA

The reproductive success of marine species with external fertilization depends on environmental conditions during gamete release. There is special interest presently in whether water motion causes sperm limitation under natural conditions. We investigated gamete release of Fucus vesiculosus from an exposed shore to ascertain: 1) when gametes are released during the tidal cycle, 2) when fertilization occurs, and 3) what the natural sperm:egg ratios are. Water samples were collected and concentrated over five minutes every half hour off Pemaquid Point, ME from three replicate sites within each of two locations using a pump-filter device. Immunofluorescence microscopy revealed that gamete release occurred only on the two calmest spring tides. Sperm became present in the water column at the same time as oogonia (30 min⁻¹ h prior to high tide [HT]) and reached peak concentration at exactly HT. The sperm:egg ratio was 76:1 on 8 Oct 1999 and 21:1 on 8 Nov 1999 at exactly 30 min prior to HT and dropped sharply after HT. Gametes continued to be collected for several hours after HT but analysis of pronuclear position in aceto-iron-hematoxylin stained eggs revealed that all fertilization occurred at approximately HT. We modelled the total number of days when reproduction was possible using these results and wind and wave data from the National Data Buoy Center. Our research provides evidence that gamete release by F. vesiculosus occurs at slack HT on calm days and that sperm are not a limiting factor in fertilization for this species.