Study of the sexual maturity of female bluefin tuna: purification and partial characterization of vitellogenin and its use in an enzyme-linked immunosorbent assay

Plasma steroid levels of female bluefin tuna (BFT) Thunnus thynnus rose from c. 1·5 ng ml⁻¹ during the quiescent period (March) to c. 7 ng ml⁻¹ during the ripening period (May). Testosterone (T) increased further to c. 8 ng ml⁻¹ during the pre-spawning period (June) while 17β-oestradiol (E₂) began to decrease. In the post-spawning period (August) steroid levels decreased to < 1 ng ml⁻¹. Vitellogenin (Vtg) plasma levels seemed to follow changes in E₂, showing an increase from the quiescent period to the ripening period of c. 18 mg ml⁻¹, decreasing slightly before spawning, and then decreasing after spawning. The Vtg content in plasma showed a good correlation both with the plasma levels of E₂ and T and with the percentage of vitellogenic oocytes at different periods of the reproductive cycle. Thus the ELISA could be taken as validated. Immunohistochemical staining of ovaries with anti BFT-Vtg serum demonstrated a high cross-reactivity with yolk proteins allowing the identification of vitellogenic oocytes.     

Reproductive characteristics of the male herring in the northern Baltic Sea

The gonad weight and gonadosomatic index of the male Baltic herring Clupea harengus membras L., were highest at the beginning of the reproductive season (April/May), the values decreasing towards the end of it (July/August) during 1988–1991. The decline could not be explained by fish size but may have been due to fish condition. A high individual variation was typical for both gonad weights and gonadosomatic indices in fish of the same size and maturity stage. The mean density of sperm cells was significantly higher in June (34·9 × 10⁹ ml⁻¹) than in July (19·2 × 10⁹ ml⁻¹, Mann-Whitney U= 17; P<0·05), the variation among the males being high in both groups. Electron microscope analysis showed a severe disruption of the mitochondrial elements in males spawning at 22°C.     

Plasma cortisol and 17β-oestradiol levels in roach exposed to acute and chronic stress

Plasma cortisol levels were measured as an indicator of physiological stress in roach subjected to brief handling, or to a 14-day period of confinement, and in undisturbed control fish, during winter (water temperature 5° C) and summer (16° C), at which time plasma 17 β-oestradiol levels were also determined. Cortisol levels in undisturbed roach were low (mean 8·1 ng ml⁻¹ at 5° C; 1·4 ng ml⁻¹ at 16° C) and both handling and handling+confinement elevated blood cortisol levels significantly to 400 and 140 ng ml⁻¹, respectively (at 5° C) and 700 and 600 ng ml⁻¹, respectively (at 16) C). Blood cortisol levels had almost returned to baseline within 4 h following handling alone but in fish subjected to handling and prolonged confinement cortisol levels remained elevated for up to 168 h. Differences in baseline and poststress levels of cortisol, and in the rate of recovery from acute stress, were observed at the two different temperatures and the possible factors underlying these differences are discussed. Circulating levels of 17 β-oestradiol were reduced significantly within 24 h of exposure to either acute handling or chronic confinement indicating that the reproductive endocrine system in roach is sensitive to disruption by stressors.     

Sequence of gonadal events and oestradiol levels in Sparus aurata (L.) under two photoperiod regimes

Individually tagged Sparus aurata were kept in tanks with running sea water (21°± 2°C) during their second and third years of life. Gonads were biopsied and blood was sampled at monthly intervals. Thirteen of 50 fish in the second year and 24 of 35 fish in the third year were phenotypically females. Fish kept under 16L/8D photoperiod from July 1981 to August 1982 did not reach maturation; waves of initial ovarian growth alternated with waves of atresia. When photoperiod was shortened as from August 1982, gonadal development commenced within a month and proceeded at a rate higher than that of the control fish reared under natural photoperiod. Under natural photoperiod oestradiol serum levels (E₂) were relatively low during the resting phase, May-October (108 ± 11 pg ml⁻¹), and during the late vitellogenic phase (502 ± 76 pg ml⁻¹). High levels (1669 ± 312 and 1240 ± 172pg ml⁻¹) occurred during the early vitellogenic and the maturational phases. The high level of E₂ during the spawning season of S. aurata is explained by earlier reports indicating a prolonged breeding season with daily release of eggs, and alternating daily surges of E₂ and 17α, 20β, dihydroxy-4-pregnen-3-one, possibly a 'maturational progestin' in this fish. In phenotypic males, E₂ was highest during early spermatogenic phases (745 ± 142pg ml⁻¹) but was low (155 ± 11 pg ml⁻¹) in males with running sperm.     

Survival of Escherichia coli O157 : H7 in yoghurt during preparation and storage at 4°C

Cow's milk was inoculated with ca 10³ and 10⁷ cfu ml⁻¹ Escherichia coli O157 : H7. After fermentation at 42°C for 0–5 h, the yoghurt was stored at 4°C. Two kinds of yoghurt were used : traditional yoghurt (TY), made with Streptococcus thermophilus and Lactobacillus bulgaricus starter cultures, and 'bifido' yoghurt (BY), made with the two starter cultures plus Bifidobacterium bifidum . After 7 d E. coli O157 : H7 decreased from 3·52 to 2·72 log₁₀ cfu ml⁻¹ and from 7·08 to 5·32 log₁₀ cfu ml⁻¹ in TY, and from 3·49 to 2·73 log₁₀ cfu ml⁻¹ and from 7·38 to 5·41 log₁₀ cfu ml⁻¹ in BY. The pH values of yoghurt dropped from 6·6 to 4·5 and 4·4 in TY (for low and high pathogen inocula, respectively), and from 6·6 to 4·6 and 4·5 in BY (for low and high pathogen inocula, respectively).     

Some aspects of the biology of a cyprinid, Aspius vorax Heckel

Scales were used for the determination of age with back-calculation of length. The oldest fish was VII + years old. Back-calculation did not exhibit Lee's phenomenon. The most rapid growth occurred in summer at water temperatures over 25°C. The growth in weight was c . 331 g year^-1 after IV vears of life. Growth was well described by von Bertalanffy equation : l_l - 91·0 [ l= ^{0·122(l0·25)}] The length-weight relationship followed the cube law (b = 3·0601) K_n ranged from 0·74 to 1·18 with a mean value of 1·0. Spawning occurred in January, fecundity was 74 509 with a mean of 1157 eggs -¹ body weight. Mean diameter of eggs was 1071 (pm). A developed ovary had ova of two sizes, immature oocytes and mature ova. The fish is a carnivorous feeder.     

The response to capture and confinement stress of plasma cortisol, plasma sex steroids and vitellogenic oocytes in the marine teleost, red gurnard

Reproductively active female red gurnard Chelidonichthys kumu were captured on long-lines, and placed in confinement tanks for 24, 48, 72 and 96 h to examine the effect of capture and confinement on reproductive parameters (experiment I). Plasma cortisol at the time of capture was elevated to levels typical of stressed fish in other species (53–125 ng ml⁻¹). Final plasma cortisol levels in red gurnard confined for any length of time were not significantly different from one another (ranging from 17 to 43 ng ml⁻¹), indicating that fish were chronically stressed when held in captivity for up to 96 h after capture. When initial and final plasma cortisol levels were compared within confinement groups, cortisol decreased significantly after 24 and 96 h of confinement indicating that some acclimation to captivity may have occurred. In contrast, plasma 17β-estradiol (E₂) and testosterone (T) levels decreased significantly to levels comparable to those in post-spawned fish, after any period of confinement, and remained low throughout the experiment. Another group of fish was captured and confined in the same manner as experiment I but subjected to repeated blood sampling every 24 h, until 96 h post-capture. In these fish, plasma cortisol levels decreased significantly from 127 ng ml⁻¹ after 24 h confinement and thereafter showed no change (25–45 ng ml⁻¹). Plasma E₂ decreased significantly after 72 h of confinement while plasma T showed no change from levels at capture. Increased amounts of follicular atresia were found in vitellogenic oocytes of fish confined for longer periods of time in experiment I, indicating that capture and confinement stress affect reproduction negatively in captive wild fish.     

Patterns of oocyte growth, vitellogenin and gonadal steroid concentrations in greenback flounder

The pattern of oocyte development in association with changes of plasma concentrations of vitellogenin (Vtg), 17β‐oestradiol (E₂) and testosterone (T) was investigated in maturing female greenback flounder Rhombosolea tapirina over the first part of a reproductive season (February to June). Examination of oocyte size‐frequency distributions showed that the oocyte developmental pattern in R. tapirina is multiple group synchrony, and that reproductively mature fish were present at all sampling times. There were no significant temporal variations in the gonado‐somatic index ( I _G), hepato‐somatic index ( I _H), or plasma concentrations of Vtg, E₂ and T during the sampling period, which indicates that reproductive development is not synchronized within the population. Significant increases in I _G, I _H and plasma concentrations of Vtg, E₂ and T, however, were observed in vitellogenic fish, and in fish undergoing final maturation. A positive relationship was also found between the growth of oocytes and plasma concentrations of Vtg, E₂ and T, although the patterns of increase were different for each variable. Plasma concentrations of Vtg and E₂ rose steadily across oocyte sizes from 100 to 450 μm, but the rate of increase of plasma E₂ was slower than that of Vtg, and both reached a saturated concentration at oocyte sizes of c . 450 μm. In contrast, plasma concentrations of T showed no marked increase until oocytes grew beyond 400 μm.     

Growth and physiological changes during metamorphosis of Senegal sole reared in the laboratory

The metamorphosis of Solea senegalensis was studied in larvae reared at 20° C and fed four different feeding regimes. A, Artemia (4 nauplii ml⁻¹); B, Artemia (2 nauplii ml⁻¹); C, mixed diet (2 nauplii ml⁻¹ and 3 mg ml⁻¹ microencapsulated diet); and D, microencapsulated diet (3·7 mg ml⁻¹). Rotifers were also supplied in all cases during the first days of feeding. These feeding regimes supported different growth rates during the pre-metamorphosis period (regime A, G=0·376 day⁻¹; regime B, G=0·253 day⁻¹; regime C, G=0·254 day⁻¹; regime D, G=0·162 day⁻¹). Larvae started metamorphosis 9 days after hatching (DAH) when fed the regime A, 13 DAH with regime B, 11 DAH with regime C and 15 DAH with regime D. A minimum 5·6–5·9 mm L_T was required under all feeding regimes to initiate the metamorphosis. Eye translocation was completed when the larvae reached 8·6–8·7 mm L_T (regimes A, B and C), but only 7·3 mm L_T with regime D. 4·4–6·2 days were required to complete eye migration under the regimes A, B and C, and 18·3 days under the regime D. This transformation is concomitant with changes in body reserves, and with the pattern of some digestive enzymes.     

Effect of high-temperature, short-time (HTST) pasteurization on milk containing low numbers of Mycobacterium paratuberculosis

The efficacy of high-temperature, short-time (HTST) pasteurization (72 °C/15 s) when low numbers (≤ 10³ cfu ml ⁻¹ ) of Mycobacterium paratuberculosis are present in milk was investigated. Raw cows' milk spiked with Myco. paratuberculosis (10³ cfu ml⁻¹, 10² cfu ml⁻¹, 10 cfu ml⁻¹, and 10 cfu 50 ml⁻¹) was subjected to HTST pasteurization using laboratory pasteurizing units. Ten bovine strains of Myco. paratuberculosis were tested in triplicate. Culture in BACTEC Middlebrook 12B radiometric medium detected acid-fast survivors in 14·8% and 10% of HTST-pasteurized milk samples at the 10³ and 10² cfu ml⁻¹ inoculum levels, respectively, whereas conventional culture on Herrold's egg yolk medium containing mycobactin J detected acid-fast survivors in only 3·7% and 6·7% of the same milk samples. IS900-based PCR confirmed that these acid-fast survivors were Myco. paratuberculosis . No viable Myco. paratuberculosis were isolated from HTST-pasteurized milk initially containing either 10 cfu ml⁻¹ or 10 cfu 50 ml⁻¹.     

Xylanolytic activity of commercial juice-processing enzyme preparations

Of 22 commercial juice-processing enzyme preparations investigated, Clarex ML was found to exhibit the highest xylanase activity. The xylanase from Clarex ML was most active at 50–60°C and pH 5·0–5·5. The K _m and V _max values of the enzyme with oat-spelt xylan as the substrate were 8·6 mg ml⁻¹ and 42 μmol xylose l⁻¹ min⁻¹, respectively. Xylobiose was the main product of enzymatic hydrolysis of xylan.     

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.     

Long-term ammonia exposure of turbot: effects on plasma parameters

Turbot juveniles were exposed to four ammonia concentrations [0·17 (L), 0·34 (M), 0·73 (MH) and 0·88 (H) mg l⁻¹ NH₃-N] for different exposure durations (28 days minimum to 84 days). Their physiological status and growth performances were compared to a control group [0·004 (C) mg l⁻¹ NH₃-N]. No growth was observed in the H group, and by day 57, mass increase in the MH group was only 15% of that in group C. During the first month growth in the L group was similar to that in control group while it was lower (33%) in the M group; afterwards the L and M groups had a similar growth (half that of controls). Accumulation of total ammonia nitrogen (TA-N) in plasma was dependent on ambient ammonia concentrations. Plasma urea levels in ammonia-exposed fish were lower, similar or greater than in controls (depending on ammonia concentration or exposure duration). Osmolarity, Cl^– and Na⁺ plasma concentrations were stable in the L and M groups. The increases in Na⁺, Cl^–, K⁺ and total Ca concentrations observed by the end of the experiment in the H and MH groups suggest that fish failed to adapt. There was an initial rise in plasma cortisol in all ammonia-exposed groups followed by a return to basal level (1·7–4 ng ml⁻¹) in the L and M groups. In group MH, plasma cortisol peaked at 42 ng ml⁻¹ by day 14, and after a decline at c . 1 month (14 ng ml⁻¹), it rose again.     

An evaluation of the use of 4-methylumbelliferyl-β-D-glucuronide (MUG) in different solid media for the detection and enumeration of Escherichia coli in foods

The use of 4-methylumbelliferyl-β- D -glucuronide (MUG) in different solid media for the detection and enumeration of Escherichia coli in foods was evaluated by testing the effects of different substrate concentrations (50 or 100 μg ml⁻¹), incubation temperatures (37 or 41·5°C) and incubation times (8, 12, 24 and 48 h). Different kinds of foods, both naturally and artificially contaminated, were analysed. The use of selective media without differential substances and an incubation time of 24 h seem to be worthy of recommendation. In this case an incubation temperature of 37°C would be preferred and the MUG concentration could be reduced to 50 μg ml⁻¹. Incubation times shorter than 24 h, which may cause a loss of sensitivity, require higher incubation temperatures (41·5°C) and MUG concentration (100 μg ml⁻¹).     

Plasma levels of C18-, C19- and C21-steroids in captive and feral female sea bass

Morphometric analysis of the gonads of sea bass Dicentrarchus labrax revealed that captive fish matured 1 month later than feral fish, but levels of gonadal steroids were identical in both groups at the same stage of sexual development. 17β-oestradiol (E₂) (up to 3 ng ml-1) and testosterone (T) (up to 4 ng ml-1) were highest during the gametogenetic period while 17,20β,21-trihydroxy-4-pregnen-3-one (17,20β,21-P) (free and sulphated) were maximal during the spawning period. Free 17,20β-dihydroxy-4-pregnen-3-one (17,20β-P) was very low and did not change (c. 0·5 ng ml⁻¹) while 17,20β-P-sulphate increased during the spawning period in both groups (up to 2 ng ml⁻¹). In contrast cortisol levels were higher in captive fish and increased during the spawning period (up to 100 ng ml⁻¹). These results suggest that captivity delays vitellogenesis and spawning in sea bass without affecting the final levels of the gonadal steroids and further indicates a role for cortisol in the latter period. The increased levels during the spawning period suggests a pheromonal role for 17,20β-P-sulphate and 17,20β,21-P-conjugates and the involvement of 17,20β,21-P in final ooccyte maturation.     

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.     

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.     

Biochemical characterization of pectate lyases produced by fluorescent pseudomonads associated with spoilage of fresh fruits and vegetables

An improved method for purification of pectate lyases (PLI and PLII) from culture fluids of Pseudomonas fluorescens CY091 and Ps. viridiflava PJ-08-6 by using a phosphocellulose cation exchanger was described. Analysis of purified PLI and PLII by sodium dodecyl sulphate-polyacrylamide and isoelectric focusing gel electrophoresis revealed that both enzymes had been purified to near homogeneity. Optimal Ca²⁺ concentration required for PLI and PLII activity was determined to be 0·5 mmol l⁻¹. The Ca²⁺ requirement could not be replaced by other metal cations such as Mg²⁺, Cu²⁺, Zn²⁺, Fe³⁺ and Co²⁺. Optimal pH for activity was determined to be between 8·5 and 9·0. The K _m values for sodium polygalacturonate were 1·28 and 1·11 mg ml⁻¹ for PLI and PLII, respectively. Both PLI and PLII were stable at low temperatures (25°C or below) for at least 1 month. However, at 37°C, the activity decreased 50% in 36 h. Optimal temperatures for activity were estimated to be 46° and 52°C for PLI and PLII, respectively. Thermal stability of both enzymes at elevated temperatures (48°C or higher) increased when CaCl₂ or a positively charged molecule such as polylysine was present, but decreased when polygalacturonate or a negatively charged molecule such as heparin was present. PLI and PLII exhibit differential degrees of sensitivity to group-specific inhibitors, including iodoacetic acid and diethylpyrocarbonate. This result suggests that both sulphydryl and imidazole groups are important for the catalytic function of PLI and PLII.     

Flow cytometric analysis of Lactobacillus plantarum to monitor lag times, cell divisionand injury

Flow cytometry in combination with fluorescent molecular markers 5- (and 6-)carboxyfluorescein succinimidylester (CFSE) and propidium iodide (PI) have been applied todetermine lag times, numbers of cell divisions and injury after mild heat (50°C, 5 min) andnisin treatments (0·1 and 1·0 μg ml⁻¹) of Lactobacillus plantarum. Initial labelling with covalently bound dye CFSE (20 and 100 μg ml⁻¹)allowed determination of lag times and cell proliferation for up to eight generations.Double-labelling with CFSE and PI (5 μg ml⁻¹) provided additional informationabout damage levels and distributions within populations. Subpopulations surviving treatmentcould be identified easily and selectively sorted.     

Glucosyltransferase activity of commercial juice-processing enzyme preparations

Of various commercial enzyme preparations examined, Cytolase M102 was found to contain the highest glucosyltransferase activity (55 U ml⁻¹). It rapidly converted maltose to panose (Glcα1 → 6Glcα1 → 4Glc) with a V _max value of 5·8 mmol l⁻¹ min⁻¹ at 50°C in 0·05 mol l⁻¹ sodium acetate buffer (pH 4·4). The K _m value of the enzyme for maltose was 750 mmol l⁻¹. Yields of panose and glucose after 45 min of reaction, for example, were 47·2% and 52·8%, respectively, on the basis of the amount of maltose consumed.