Insemination, fertilization and gamete management in tench, Tinca tinca (L.)

Various procedures for artificial insemination in tench, Tinca tinca (L.) were re-examined with evaluation of fecundity of males and females among different tench strains. The objectives of this study were to enhance fertilization and hatching rates through optimization of the activation solution, the insemination process, the activation of gametes, and the elimination of eggs stickiness. Sperm for all experiments was collected directly into immobilization solution of modified Kurokura solution containing 180 mM of NaCl and stored at 2 °C for 2.5–5 h prior to the experiment. When dechlorinated tap water was used for activation a gamete ratio of 1150 spermatozoa per egg showed the best significant fertilisation and hatching rates. Optimal ratio between eggs (weight in g) and activation solution (in cm³) was 1:1. Different concentrations of activation solutions such as NaCl from 0 to 68 mM (0–136 mOsmol kg⁻¹) without buffer statistically decreased fertilization and hatching rates. The activation solution containing 17 mM of NaCl, 10 mM Tris–HCl, pH 8 and 9 significantly increased fertilization and hatching rates compared to dechlorinated tap water of pH 7 or activation solution containing 17 mM of NaCl, 10 mM Tris–HCl, pH 6 and 7. Adhesiveness of the eggs was successfully removed by incubation in Alcalase and activity: 3.16 Anson units per cm³.     

Kurokura Solution as Immobilizing Medium for Spermatozoa of Tench (Tinca tinca L.)

This study tested KUROKURA solution (Kurokura et al., 1984, Aquaculture 37, 267–274) and its modifications (by increasing NaCl content to 160, 180 and 200 mM) on immobilizing properties for sampling and short-term preservation of potential motility of tench spermatozoa. The immobilizing solution is used because, when collected, the sperm of most samples is contaminated by urine, causing spermatozoa to be of poor quality, with low motilities and velocities (almost 0), thus resulting in a worsened fertilization and hatching rate. Sperm was sampled with a syringe containing an immobilizing solution (IS), allowing an IS:sperm ratio of 2:1, under aerobic conditions at 0–4°C. This sperm solution was stored for 10 h and untreated sperm was collected prior to fertilization as a control. Spermatozoa quality was evaluated for the cell motility and velocity parameters and also for fertilization ability and hatching rate. Results obtained for tench sperm motility, velocity, fertilization and hatching rate showed that only sperm collected in the various immobilizing solutions can be successfully used for artificial insemination and preservation after 10 h at 0–4°C. The best immobilizing solution was found to be KUROKURA 180 (180 mM NaCl, 2.68 mM KCl, 1.36 mM CaCl₂· 2H₂O and 2.38 mM NaHCO₃), giving a fertility and hatching rate of 41%, with no change in rates after 10 h storage of sperm. Control sperm without immobilizing solution showed a fertilization and hatching rate of only 6–7%.     

Ionic composition and osmolality of paddlefish (Polyodon spathula, Acipenseriformes) seminal fluid

Changes in ionic composition as Na⁺,K⁺, Ca²⁺ and Mg²⁺, osmolality inseminal fluid, percentage of motile spermatozoaand velocity were investigated in response toCPP and different dosage of LHRHa. The lowestvelocity of sperm was observed after use CPPtreatment. The velocity of spermatozoa,significant main effect of the treatment(P < 0.0001) and the time of sperm collection(P < 0.0104) were evaluated. The osmolality ofseminal fluid was different betweenexperimental groups of LHRHa (48.0–62.7mOsmol.kg^–1) and CPP (33.0–46.3mOsmol.kg^–1) treatments. The osmolalitywas significantly higher on the first day andone-half, then declined on day three, rangingfrom 33.0 to 62.7 mOsmol.kg^–1. Analysisof variance showed significant main effects ofthe treatment (P < 0.0001) and the time ofsperm collection (P < 0.0002) on the osmolalityof seminal fluid. The level of Na⁺ andK⁺ ion was different between experimentalgroups of LHRHa and CPP treatment. The highestconcentration of 11.11 mmol.l^–1 wasobserved at Na⁺ ion. Then theconcentrations declined on the level 1.56, 0.52and 0.36 mmol.l^–1 for K⁺, Ca²⁺and Mg²⁺ ions, respectively. There werehighly positive correlations between osmolalityof seminal fluid and dosage of LHRHa treatment(r = 0.84), velocity of spermatozoa andosmolality of seminal fluid (r = 0.57) andosmolality of seminal fluid and Na⁺concentration at seminal fluid (r = 0.70).Injection with LHRHa increased quality of spermas velocity of sperm, level of Na⁺,K⁺ and osmolality at seminal fluidcompared to CPP treatments.     

Effects of ultra-violet irradiation on sperm motility and diploid gynogenesis induction in large yellow croaker (<Emphasis Type="Italic">Pseudosciaena crocea</Emphasis>) undergoing cold shock

Large yellow croaker, Pseudosciaena crocea, exhibit sexually dimorphic growth, with females growing faster and reaching larger adult sizes than males. Thus, development of techniques for preferentially producing females is necessary to optimize production of these species. We have established a protocol to produce all-female croaker P. crocea through induction of meiotic gynogenesis with homologous sperm. The first set of experiments investigated the ultra-violet (UV) irradiation on sperm motility and duration of sperm activity to determine the optimal UV dosage for genetic inactivation of sperm, yet retaining adequate motility for activation of eggs. Milt from several males was diluted 1:100 with Ringer’s solution and UV irradiated with doses ranging from 0–150 J cm⁻². The results indicated that motility and duration of activity generally decreased with increased UV doses. At UV doses greater than 105 J cm⁻², after fertilization, motility was <10% and fertilization rates were significantly lower. Highest hatching rate was obtained at 75 J cm⁻². A second set of experiments was carried out to determine appropriate conditions of cold shock for retention of the 2nd polar body in P. crocea eggs after fertilization with UV-inactivated sperm by altering the timing, temperature and duration of shock. At 20°C, shock applied at 3 min after fertilization resulted in higher survival rate of larvae at 6 h after hatching. Results of different combinations of three shock temperatures (2°C, 3°C or 4°C) and five shock durations (4 min, 8 min, 12 min, 16 min or 20 min) at 3 min after fertilization demonstrated that shocks of 12 min gave highest production of diploid gynogens. Statistical analysis revealed that maximum production of diploid gynogens (44.55 ± 2.99%) were obtained at 3°C. The results of this study indicate that the use of UV-irradiated homologous sperm for activation of P. crocea eggs and cold shock for polar body retention is an effective method for producing gynogenetic offspring.     

The teleost pseudobranch: a role for preconditioning of ocular blood supply?

The physiological relevance of the teleost pseudobranch as a remnant of a reduced gill arch is still unclear. Numerous hypotheses have been proposed regarding its physiological role, but direct confirmatory evidence is lacking. The close relationship by serial blood flow arrangement with the fish eye’s choroid rete has sparked the idea that pseudobranchial preconditioning of blood pH may facilitate initiation of the Root effect and thus support the establishment of high oxygen tensions for retinal diffusive supply. This idea was critically tested by studies on isolated pseudobranchs in situ (Oncorhynchus mykiss), perfused with RBC/Ringer or RBC/plasma suspensions of widely varied composition (pH 7.4–8.2). Detailed analysis of inflowing as compared to effluent perfusates indicated normal aerobic metabolism expressed by a rise in Pco₂ (+0.39 ± 0.13 mmHg ), an oxygen utilization of 25% and a high oxygen consumption of ∼400 nmol g⁻¹ min⁻¹. Upon passage of the pseudobranch, pH (corrected for Haldane effect) was only slightly acidified (−0.03 to −0.10), [HCO₃ ⁻] and [lactate] were slightly enhanced (+0.51 mmol l⁻¹ or 0.13 mmol l⁻¹, respectively). In order to test for yet unknown plasma components involved in pseudobranch function, a second series of experiments was conducted using RBC-suspensions in fresh plasma instead of Ringer, with results closely resembling those of the RBC/Ringer series. Lacking any physiologically significant correlation with the level of perfusate pH, the obtained data indicate pseudobranchial basic metabolic activity rather than pH regulatory characteristics. Also the observed absolute changes in pH are negligible in terms of pH regulation towards the Root-threshold. Accordingly, the present experiments as well as plausibility evaluation of mechanisms do not support the idea of blood pH pre-adjustment prior to entry into the choroid rete structure of the teleost eye to facilitate the Root-mediated oxygen release.     

Studies on sperm of diploid and triploid tench, Tinca tinca (L.)

The tench Tinca tinca is an interesting fish from the viewpoint of polyploidy and related atypical reproduction aspects. Triploid tench were produced artificially. Studies of spermiation as well as of sperm motility and structure were performed on several triploid and diploid males simultaneously with individual experimental crosses with diploid females to define their reproductive capacities. The testes of triploids visually looked less developed in the most of cases with lower sperm production (0.05 cm³ sperm per male), GSI and weight of testes compared to diploids (0.58 cm³ sperm per male). Analysis of variance showed significant influence of ploidy level on the percentage of motile spermatozoa. Triploidy did not change percentage of live spermatozoa and velocity of spermatozoa at the first time of sperm movement. The study of sperm structure by scanning electron microscopy revealed that most sperm cells were of normal structure with some anomalies. Sperm heads of triploid and diploid males were mostly round-shaped, 1.86±0.2 and 1.6±0.18 μm in diameter. The midpiece of triploid spermatozoa was slightly narrower than that of diploid ones with typical cylindrical shape. Flow cytometry revealed sperm cells of triploids to be largely aneuploid (1.47 n) with high mosaic DNA, oscillating from haploid DNA content (1.0 n) to diploid DNA content (1.9 n). Experimental crosses between triploid males and diploid females revealed that these males were capable to stimulate effective development with relatively high level of fertilization and hatching rates from 0 to 70%. In conclusion, triploidization does not seem to guarantee sterility of tench.     

Physio-Chemical Characteristics of Seminal Plasma and Development of Media and Methods for the Cryopreservation of European eel Sperm

The high sperm density, together with the short spermatozoa swimming time, makes European eel sperm manipulation and assessment for quality difficult. Two diluting media (K15 and K30) previously designed for Japanese eel sperm were tested. After 24 h, European eel sperm showed significant reduction in the percentage of motile spermatozoa after activation and different motility parameters (VAP, angular velocity; VCL, curvilinear velocity; VSL, straight line velocity; BCF, beating cross frequency), concluding that these media are not suitable to preserve the sperm of this species. After a hormonal treatment to induce spermiation, sperm volume, density and motility were recorded at weekly samplings. The variation of the osmolality (325–330 mOsm kg⁻¹), pH (8.4–8.6) and the ionic composition (concentration of Na⁺, K⁺, Mg²⁺ and Ca²⁺) of the seminal plasma were registered. Physio-chemical results were related with sperm quality throughout the treatment, to determine which must be the suitable characteristics of one extender for the sperm of this species, and to find the best conditions to obtain suitable cryopreservation media for European eel sperm. K⁺ concentration increased, while Ca²⁺ and Mg²⁺ concentrations showed a progressive reduction in correlation with the sperm quality improvement. Na⁺ showed a decreasing, but not significant tendency. P1 and P2 freezing media were designed considering the physio-chemical parameters as well as the ionic composition shown by the best quality sperm samples, and then compared with the previously described solutions, TNK and K30. Sperm quality was determined, checking the percentage of motile spermatozoa and motility parameters using computer-assisted sperm analysis (CASA) software. Samples were frozen after dilution (1:5, 1:20, 1:100) in different freezing media supplemented with 10% dimethyl sulfoxide (DMSO). After thawing, samples frozen with low dilution ratio (1:5) in TNK and P1 media showed higher, although not significant, spermatozoa survival (35.5 ± 14.5 and 36.6 ± 6.7%). The addition of l-α-phosphatidylcholine to the media seems to have a positive effect, as reported in the Japanese eel.     

The effect of luteinizing hormone-releasing hormone analogue (LHRHa) on sperm count of Oreochromis niloticus (L.)

An experiment was conducted to compare the potency of different levels of luteinizing hormone-releasing hormone analogue (LHRHa) for increasing the sperm counts in sexually mature male Oreochromis niloticus (L.). One hundred males (106.49 ± 3.59g, 15.2 7 ± 0.20 cm) were randomly distributed in four treatment groups, each receiving intramuscular injections of: (i) control, 0.05 ml phosphate-buffered saline, (ii) 10 μg kg^-1 LHRHa, (iii) 20 μ kg^-1 LHRHa and (iv) 30 μ kg^-1 LHRHa. Sperm counts were determined prior to injection (day 0) and for four consecutive days (days 1 to 4) thereafter. Results showed a significant increase in sperm counts (P < 0.01) 1 day after injection among males injected with 10 to 20 μ kg^-1 LHRHa. The effect of injecting 30 μ kg^-1 LHRHa is similar to that in the control group. Across all treatments, sperm counts declined with time over the 4-day sampling period.     

Biochemical characterization of Siberian sturgeon Acipenser baeri and sterlet Acipenser ruthenus milt plasma and spermatozoa

This paper provides data concerning biochemical composition of milt of two sturgeon species, Siberian sturgeon bred in aquaculture facility of Inland Fisheries Institute in North Poland and sterlet (from two different populations from Danube and Odra). Milt plasma of Siberian sturgeon and sterlet, when compared to teleost fish, is characterized by much lower osmolality (up 70 to 96 mOsm kg⁻¹) and protein concentration (0.24–0.58 g l⁻¹). In spite of the presence of an acrosome and acrosin the antiproteinase activity of seminal plasma was low (12.79–25.40 U l⁻¹). Activities of arylsulfatase and β-N-acetylglucosaminidase were found in spermatozoa. This agrees with the presence of an acrosome in sturgeons sperm. Similarly to mammals, these enzymes are also present in milt plasma. We determined a range of enzymatic activities from the minimal (seminal plasma) to the maximal damage (supernatants obtained after freezing-thawing without cryoprotectant). Activities of arylsulfatase, β-N-acetylglucosaminidase, lactic dehydrogenase and acid phosphatase were released from spermatozoa after freezing-thawing. For this reason they are good potential candidates as a markers of cryoinjury to sperm acrosome and midpiece. This revised version was published online in July 2006 with corrections to the Cover Date.     

Tolerance of striped trumpeter Latris lineata embryos to ozonated seawater

The tolerance of striped trumpeter, Latris lineata (Bloch and Schneider 1801) embryos to ozonated seawater was examined as a possible means of disinfection. The effect of a range of ozone doses and exposures (CT = concentration × exposure time) was tested at different stages of embryonic development. Three-day-old embryos two-thirds developed around the yolk were exposed for 0.5, 1 or 5 min to ozone concentrations of 0.5, 1, 2 and 5 mg O₃ l⁻¹ in a fully orthogonal factorial design. For each treatment there were four replicate 250 ml containers that each received 100 ± 15 embryos. There was no significant difference in hatching success between control-treated embryos or embryos ozonated at 0.5 or 1 mg O₃ l⁻¹ for 0.5, 1 or 5 min (P < 0.05). However, hatching success was significantly reduced when embryos were treated with 2 or 5 mg O₃ l⁻¹ for 5 min or 5 mg l⁻¹ O₃ for 1 min (P < 0.05). The tolerance of embryos to 0, 0.5 or 2 mg O₃ l⁻¹ for 1 min at different stages of development (Day 0, 1, 2, 3 or 4), was then examined. An ozone concentration of 0.5 mg l⁻¹ had no effect on hatching success at any stage of development, but a concentration of 2 mg l⁻¹ significantly reduced hatching success on all days except Day 3. A safe and tested hatchery practise is to disinfect striped trumpeter embryos with 1 mg O₃ l⁻¹ for 1 min on Day 3 post-fertilisation when the embryo is two-thirds developed around the yolk.     

The effect of feeding on oxygen consumption and ammonia excretion of juvenile tench Tinca tinca (L.) reared in a water recirculating system

The effect of feeding frequency (one, three, and continuous feeding), feed ration (0.2, 0.5, 0.8% of total fish biomass), and feeding per se on the oxygen consumption (OC, mg O₂ kg⁻¹ h⁻¹) and ammonia excretion (AE, mg TAN kg⁻¹ h⁻¹) of juvenile tench (body weight 15–19 g) and variations in these parameters in daily cycles were examined. Fish metabolism was studied in a recirculating system (rearing tanks of 0.2 m³, water temperature 23 °C). It was found that oxygen consumption and ammonia excretion depended significantly on feed ration. An increase of feed ration from 0.2 to 0.8% of fish biomass caused an increase of OC and AE from 126.80 mg O₂ kg⁻¹ h⁻¹ and 1.95 mg TAN kg⁻¹ h⁻¹ to 187.35 mg O₂ kg⁻¹ h⁻¹ and 8.80 mg TAN kg⁻¹ h⁻¹ (p<0.05). There was no dependence between feeding frequency and the mean rate of oxygen consumption. However, the relationship between feeding frequency and ammonia excretion by juvenile tench was statistically significant (p<0.05). Feeding frequency significantly affected daily fluctuations of AE and OC. It was found that diurnal variations in metabolic rates were strictly related to tench feeding, and the daily variations of AE were significantly higher than OC.     

Minimally invasive surgical technique for egg collection from the Persian sturgeon, Acipenser persicus

A minimally invasive surgical technique (MIST) for the removal of ovulated eggs from Persian sturgeon Acipenser persicus was tested on broodstocks caught from the wild to determine whether it affected fecundity or hatching rates compared with the traditional stripping method of killing and removing the eggs (commonly used in hatchery). Morphological parameters of females, germinal vesicle (GV) position, weight of obtained egg, number of eggs/gram, fertilization rate, and percentage of hatching during incubation were not significantly different between the MIST and traditional stripping methods. Obtained ova were 4.8 ± 0.4 kg female⁻¹ in the MIST and 4.6 ± 0.5 kg female⁻¹ in traditional stripped groups, respectively; the fertilization rate was 83.4 ± 11.2% and 80.0 ± 7.2% in groups, respectively. The results of this study showed that the minimally invasive surgical technique approach is efficient, practical, and less stressful to Persian sturgeon broodstocks during artificial propagation than other reported egg collection procedures.     

LHRHa‐induced ovulation of the endangered‐Caspian brown trout (Salmo trutta caspius) and its effect on egg quality and two sex steroids: testosterone and 17α‐hydroxyprogestrone

To induce synchronized ovulation, migrating wild Caspian brown trout (Salmo trutta caspius) females were treated with two interperitoneal injections of Des‐Gly¹⁰, d ‐Ala⁶ LHRH (LHRHa), given 3 days apart. Two injections of 100 μg kg^?1 body weight of this hormone effectively induced ovulation. Within 27 days from the second injection, all fish injected with 100 μg kg^?1 LHRHa had ovulated compared with 54.5% of the controls. The mean time to ovulation was reduced significantly (P<0.05) from 31.67±4.84 days in control fish and 28.83±7.31 days in sham‐treated fish to 16.36±1.61 days in fish injected with 100 μg kg^?1 LHRHa. The fertilization rate in 50 and 100 μg kg^?1 LHRHa‐injected fish was significantly lower than that in the control fish (P<0.05). In fish injected with 50 and 100 μg kg^?1 LHRHa, significant (P<0.05) changes in testosterone (T) and 17α‐hydroxyprogestrone (OHP) levels were observed. After the second LHRHa injection, the fish injected with 100 μg kg^?1 showed the highest serum levels of testosterone and OHP. These results demonstrate that the use of LHRHa can effectively reduce the mean time to ovulation and induce synchronized ovulation in Caspian brown trout.     

Pre-spawning water temperature affects sperm respiration and reactivation parameters in male carps

Concentration, ability to motility, motility during the second activation (reactivation), and endogenous respiration were studied in sperm from two experimental groups of carp males. Group 1 was maintained for 7 days at 15°C (cold water (CW) group), whereas the second group was subjected to a temperature of 20°C (warm water (WW) group) before sperm sampling. Reactivation were achieved after incubation of firstly activated sperm in media with osmotic pressure adjusted up to 300 mOsm*kg⁻¹ by increasing K⁺ concentration. Statistically significant reduction of spermatozoa concentration in CW samples versus WW (from 46.0 ± 12.5 (15°C) to 59.3 ± 7 10⁹ (20°C) spermatozoa /ml) have been observed. The sperm of the CW group required a significantly longer incubation time (37 min) under isotonic conditions to achieve a maximum percentage of potent motility at repeated activation than the WW group (23 min). After activation of sperm motility, an increase of respiration rate up to maximum level has been found, this level remained the same under condition of recovering the potential to repeated activation. During the sperm movement respiration rate, in CW group (6.1 nmolO₂/min/10⁹spermatozoa) and WW (3.9 nmolO₂/min/10⁹spermatozoa), was significant higher compared to nonactivated sperm (2.4 nmolO₂/min/10⁹spermatozoa for CW and 1.1 nmolO₂ /min/10⁹spermatozoa for WW). And keeping males for 7 days at 15°C increase the respiration rate of sperm.     

Calcium balance in embryos and larvae of the freshwater-adapted teleost,Oreochromis mossambicus

Changes in Ca²⁺ content and flux, and the development of skin chloride cells in embryos and larvae of tilapia, Oreochromis mossambicus, were studied. Tilapia embryos hatched within 96h at an ambient temperature of 26–28°C. Total body Ca²⁺ content was maintained at a constant level, about 4–8 nmol per individual, during embryonic development. However, a rapid increase in body Ca²⁺ level was observed after hatching, 12.8 to 575.3 nmol per individual from day 1 to day 10 after hatching. A significant influx and efflux of Ca²⁺ occurred during development, with the average influx rate for Ca²⁺ increasing from 5.9 pmol mg⁻¹ h⁻¹ at 48h postfertilization to 47.8 pmol mg⁻¹ h⁻¹ at 1 day posthatching. The skin was proposed as the main site for Ca²⁺ influx before the development of gills, and the increased Ca²⁺ influx may be ascribed to gradual differentiation of skin surface and chloride cells during embryonic development. Ca²⁺ efflux was 16–56 pmol mg⁻¹ h⁻¹ in 1-day-old larvae. The resulting net influx of Ca²⁺, 10–12 pmol mg⁻¹ h⁻¹, accounted for the increased Ca²⁺ content after hatching. When comparing the measured and estimated ratios of efflux and influx, active transport was suggested to be involved in the uptake of Ca²⁺. Chloride cells, which may be responsible for the active uptake of Ca²⁺, started to differentiate in the skin of embryos 48h after fertilization, and the density of chloride cells increased following the development. A possibility of active transport for Ca²⁺ in early developmental stages of tilapia is suggested.     

Spermiation of paddlefish (Polyodon spathula,Acipenseriformes) stimulated with injection of LHRH analogue and carp pituitary powder

The potential of carp pituitary powder (CPP) at one dose, or the luteinizing hormone-releasing hormone (LH-RH) analogue, des–Gly¹⁰,(d–Ala⁶)–LH-RH–ethylamide, at three different doses to stimulate spermiation in paddlefish (Polyodon spathula) was tested. Single injections of the LH-RH analogue at 0.2, 0.1, or 0.05 mg·kg^–1 increased the number of spermatozoa per kilogram of body weight (kg^–1 b.w.) by 4.7, 3.4, and 3.4 times respectively compared to control, but the number of spermatozoa per kilogram of body weight decreased with CPP (4 mg·kg^–1) by 1.7 times compared to the control. The LH-RH analogue prolonged active spermiation, with numbers of spermatozoa ranging from 7.69 to 1.19 × 10⁹ kg^–1 b.w. up to 96 h after treatment. Analysis of variance showed significant influence of experimental groups on volume of sperm per male and per kilogram of body weight, and the total number of spermatozoa per kilogram of body weight, but insignificant influence on the total number of spermatozoa per male. The percentage of motile spermatozoa was not different between experimental groups for sperm collection at different times after injection. A very high positive correlation (r = 0.93) was obtained between sperm concentration and sperm transmittance measured with a spectrophotometer. This relationship was described with the following linear regression: sperm concentration (× 10⁹ mL^–1) = 1.3244 X^–0.9969, where X is the percentage of sperm transmittance.     

Induction of ovulation in ornamental common carp (Koi, Cyprinus carpio L.) using LHRHa ([d-Ser(tBu)6, Pro9-NEt]-LHRH) combined with haloperidol and carp pituitary extract

The effects of a single injection of mammalian superactive analogue [d ‐Ser(tBu)⁶,Pro⁹‐NEt]‐LHRHa (20 μg/ kg^?1) combined with the dopamine antagonist, haloperidol (HAL, 0.5 mg kg^?1), for induction of ovulation in the koi carp broodstocks were determined under routine hatchery conditions. The results were compared with classic carp pituitary extract (CPE, double injection) application (water temperature 22 °C). Physiological saline (0.7% NaCl)‐injected fish were used as a control group and no ovulation occurred in this group. The spawning ratio was high in the LHRHa+HAL treatment group and in the CPE treatment group (6/7 and 5/7 respectively). The latency period was 14–16 h in the LHRHa+HAL treatment group and 12–14 h in the CPE treatment group (after the second injection). There was no difference between the two ovulating groups with respect to the spawning index (the weight of eggs as a percentage of female body weight) and fertilization rate of eggs (P>0.05). As a result, ovulation can be induced successfully in koi carp broodstocks with 20 μg kg^?1 [d ‐Ser(tBu)⁶,Pro⁹‐NEt]‐LHRHa+0.5 mg kg^?1 HAL treatment in a single injection without decreasing the egg quality. Application of this combination can be useful for hatchery and broodstock management in koi carp culture.     

Artificial insemination in tench, Tinca tinca L.

Abstract. The sperm of tench, Tinca tinca L., is characterized by a milky colour and consistency, and is of very low density. After collecting the sperm, motion of spermatozoa was recorded even without water activation. A better motility value (value 4·36 on average) was observed in spermatozoa collected in immobilizing solution (collecting medium) and stored for 3h, when compared with spermatozoa without collecting medium. Average total and relative numbers of spermatozoa were 12·16 × 10⁹ per male and 18·50 × 10⁹ per kg of body weight, respectively. When testing the effect of activating solution in artificial propagation of tench, the highest fertilization rates (81·3 and 85% in two cases) were found for NaCl solution with an osmotic concentration of 34 or 69 mOsmol and for fresh water, respectively. The fertility rate was reduced significantly ( P < 0·01) by any increase above 105 mOsmol in NaCl concentration in the activating solution. In the tests of optimal method of artificial fertilization, the highest hatching rate of sac fry (71·35%) was found in sperm collected into immobilizing solution. The application of immobilizing solution significantly increased the number of sac fry at the levels P < 0·1 and P < 0·01, if compared with intact sperm stored for 3 h and fresh sperm, respectively.     

Improvement of common carp artificial reproduction using enzyme for elimination of egg stickiness

This study summarizes optimization of techniques for common carp artificial propagation including improvements of activation solution (AS), the process of insemination, and elimination of egg stickiness. The optimum gamete ratio for good fertilization and hatching rate ranged from 8490 to 23 672 spermatozoa per egg, when dechlorinated tap water was used. Optimal ratio between eggs (weight in g) and AS (in ml) was defined as 1:1 to 1:2. Different concentrations of AS such as NaCl from 0 to 34 mM (0–68 mOsmol kg^–1) did not change fertilization and hatching rates. An AS adopted for carp spermatozoa (45 mM NaCl, 5 mM KCl, 30 mM Tris–HCl, pH 8) was compared with other saline AS; only the 51 mM (102 mOsmol kg^–1) NaCl solution decreased fertilization and hatching rate. The AS containing 20 mM Tris–HCl at pH 9 increased fertilization and hatching rates compared to dechlorinated tap water of pH 7 or to AS of pH 6 and 7. Adhesiveness from the eggs was successfully removed by incubation in Alcalase DX (PLN 04715) using two successive steps with different enzyme concentrations. The first step with an enzyme concentration of 2 ml l^–1 was applied from 8 to 20 min after fertilization. Later in a second step, the best time for application of alcalase enzyme at a concentration of 20 ml l^–1 was for 45 and 60 s at 20 min after fertilization leading to fertilization and hatching rates of 80–87%. The α-Chymotrypsin (EC 3.4.21.1. Merck) was also found effective for elimination of stickiness. Results with α-Chymotrypsin enzyme indicate that the response to success in elimination of stickiness is highly variable mainly due to differences in the environment, quality of water and carp strains.     

Pharmacokinetics of ivermectin in sea bream, Sparus aurata using a direct competitive ELISA

The pharmacokinetics of ivermectin in the serum of cultured sea bream, Sparus aurata, after a single intraperitoneal injection of 100 μg kg⁻¹ body weight was studied by the use of direct competitive ELISA. Pharmacokinetic analysis of the serum concentrations versus time points obtained was performed using non-comparmental analysis and a compartmental pharmacokinetic model approach. In the latter case a two-compartment open model with a lag time gave the best fitting. The maximum peak serum concentration was 308.4 ng ml⁻¹ at 2 h post treatment. The AUC of ivermectin was 10700 ng h ml⁻¹ and the elimination half-life 15.37 h, indicating a rapid uptake, high bioavailability and fast elimination of the drug by sea bream. This revised version was published online in July 2006 with corrections to the Cover Date.