Optimalisation of the Activation Medium and Effect of Inhibiting Activities of Acid Phosphatase, Lactate Dehydrogenase and ß-N-Acetylglucosaminidase on the Fertilisation Success of Eurasian Perch (Perca fluviatilis L.).

Although methods for the artificial reproduction of perch have been developed, a lack of information remains regarding the enzymes present in its semen, as well as their role in the fertilisation process. In this study, we first select the optimal activating solution for perch fertilisation and then determine the inhibition effect of enzymes that have already been reported as present in the sperm of teleosts-acid phosphatase (AcP), lactic dehydrogenase (LDH) and ß-N-acetylglucosaminidase (ß-NAGase)-on the percentage of motile spermatozoa and fertilised eggs. Of the 8 studied activation media, a solution composed of 80 mM NaCl, 20 mM KCl, 10 mM Tris, with pH 8.0 and 206 mOsm/kg proved to be optimal for perch gametes. The addition of ammonium molybdate (AcP inhibitor) caused no significant reduction in the percentage of fertilised eggs. On the other hand, the addition of 0.25 mM gossypol (LDH inhibitor) and 0.125 M acetamide (ß-N-acetylglucosaminidase inhibitor) significantly decreased the fertilisation percentage to 41.1% and 52.4%, respectively, in contrast to the control (89.9 %). Both LDH and ß-NAGase thus seem to play a very important role in the perch fertilisation process.

Dietary ARA Improves COX Activity in Broodstock and Offspring Survival Fitness of a Model Organism (Medaka Oryzias latipes).

A 3-week feeding trial was conducted in medaka broodstock (age five months) to examine the effect of dietary arachidonic acid (ARA) level (range: 4-23 mg g-1 of total fatty acids (TFAs)) on fertility, cyclooxygenase (COX) activity, egg size, sperm motility parameters, hatching rate and weight of hatch, survival and growth of larvae. After spawn induction and dietary exposure to 4 mg ARA g-1 TFA, broodstock were fed a diet containing ARA in the amounts: 4 (continued, as control), 5, 14 and 23 mg g-1 TFA. COX1 activity in the liver and the number of COX2-positive cells in the ovaries was increased in females fed the diets containing the two highest amounts of ARA. The highest sperm motility parameters were observed in males fed a diet containing 23 mg ARA g-1 TFA. The hatchability rate and bodyweight of hatchlings were higher in the group obtained from broodstock fed a diet containing 23 mg ARA g-1 TFA (79% and 0.66 mg fish-1, respectively) compared with 4 mg ARA g-1 TFA (50% and 0.40 mg fish-1). The average mortality of offspring obtained from this group at 7 days post hatching was significantly higher than that of all other groups.

Dietary resveratrol improves immunity but reduces reproduction of broodstock medaka Oryzias latipes (Temminck & Schlegel).

Here, we investigated the effect of dietary resveratrol (20, 40, and 80 µg/g BW/day) on cell-mediated immunity (activity of spleen phagocytes and proliferative response of lymphocytes) and reproductive parameters (egg and sperm quality, i.e. fecundity-total number of eggs produced by individual fish, fertility, embryo survival, and hatching rate) in medaka. Fish fed feed with resveratrol at 40 and 80 µg/g BW/day had significantly higher metabolic activity and intracellular phagocyte killing activity than control. The proliferative lymphocyte activity of the fish from R80 group was greater by more than 20 % in comparison with the control group (P < 0.05). The percentage of macrophages (MO) and their mean fluorescence intensities (MFI) in R40 and R80 groups were significantly higher compared to C and R20 groups (P < 0.05). The differences in MO and MFI values ranged from 52.5 % (±1.5; R0 group) to 65.8 % (±1.6; R80 group) and from 23.2 (±1.4; R0 group) to 38.2 (±2.4; R80 group), respectively. Moreover, resveratrol at 80 µg/g BW/day decreased liver COX activity, i.e. 5.4 in R80 group and 7.9 in R0 group (P < 0.05). The motility parameters of the sperm obtained from the males fed feed supplemented with resveratrol at 80 µg/g BW/day exhibited the highest values except the linearity, which was lower as compared to the control (P < 0.05). The results indicate that diet supplemented with resveratrol at a dosage of 40 µg/g BW/day improves phagocyte killing ability and lymphocyte proliferation in broodstock and accelerates offspring hatch. Also, the results suggest that COX activity influences sperm and oocyte quality in fish; the presence of a COX inhibitor in the dose of 40 µg/g BW/day decreased the embryo survival.

Androgenetic development of X- and Y-chromosome bearing haploid rainbow trout embryos.

Haploid fish embryos are important in studies regarding role of the recessive traits during early ontogeny. In fish species with the male heterogamety, androgenetic haploid embryos might be also useful tool in studies concerning role of the sex chromosomes during an embryonic development. Morphologically differentiated X and Y chromosomes have been found in a limited number of fish species including rainbow trout (Oncorhynchus mykiss Walbaum 1792). To evaluate role of the sex chromosomes during rainbow trout embryonic development, survival of the androgenetic haploids in the presence of X or Y sex chromosomes has been examined. Androgenetic haploid rainbow trout were produced by fertilization of X-irradiated eggs with spermatozoa derived from the normal males (XY) and neomales, that is, sex-reversed females (XX) to produce X- and Y-bearing haploids, and all X-bearing haploids, respectively. Survival rates of the androgenetic progenies of normal males and neomales examined during embryogenesis and at hatching did not differ significantly. However, all haploids died within next few days after hatching. Cytogenetic analysis of the androgenetic embryos confirmed their haploid status. Moreover, apart from the intact paternal chromosomes, residues of the irradiated maternal chromosomes observed as chromosome fragments were identified in some of the haploids. Provided results suggested that rainbow trout X and Y chromosomes despite morphological and genetic differences are at the early stage of differentiation and still share genetic information responsible for the proper embryonic development.

Quality and quantity of smelt (Osmerus eperlanus L.) sperm in relation to time after hormonal stimulation.

The effect of Ovaprim (salmon GnRH analogue and a dopamine antagonist) treatment on the quantity and quality of smelt (Osmerus eperlanus L.) sperm was studied in relation to time after hormonal stimulation. Sperm was obtained at 0, 24, 48 and 72 h after treatment (n=13/each time point). Computer Assisted Sperm Analysis (CASA) was used to evaluate sperm motility parameters and histological analysis was used to examine the testis morphology. Only a small volume of semen (1-5 µl) was collected at the beginning of the experiment (time 0) but it dramatically increased 24 h after hormonal treatment. A further increase in semen volume was recorded 48 h after hormonal stimulation. CASA parameters, such as percentage of motile cells, progressive motility, curvilinear velocity, straightlinear velocity, straightness and amplitude of lateral head displacement of stripped sperm increased 48 h after hormonal treatment, which indicates high quality of sperm. No further increase in sperm quality was recorded at 72 h. Compared to stripped semen, testicular semen was characterized by a slightly lower quality. In addition, histological analysis indicated that 24 h after hormonal treatment, a high number of spermatozoa was released from the testis. Ovaprim-stimulated smelt became clearly darker than the control fish. In conclusion, our results suggest that smelt semen should be collected 48 h after hormonal stimulation to ensure high quality of semen. This time may vary depending on maturation status of testis.

Aromatase expression in testes of XY, YY, and XX rainbow trout (Oncorhynchus mykiss).

The main goal of the present study was to show whether testicular cells of rainbow trout (Oncorhynchus mykiss Walbaum) either hormonally manipulated (XX males) or produced by using gamma irradiation and pressure shock (YY males, "supermales") are able to aromatize androgens into estrogens compared with the control (XY males). The expression of aromatase gene at the level of the protein and its presence in testicular tissue was investigated by means of immunohistochemistry and Western blot analysis, respectively. The positive staining for aromatase was detected in testicular cells of all trout and in efferent duct cells of XY and YY males. However, the staining intensity varied among particular trout, being strong in YY males, moderate in XY males, and weak in XX trout. It was confirmed by quantitative image analysis in which the staining intensity was expressed as relative optical density (ROD) of diaminobenzidine deposits. Significant differences were found between XY and YY trout ((**)p<0.01) and XY and XX trout ((*)p<0.05). Such differences could reflect various levels of estrogens, possibly dependent on the genetic background of the trout studied. It seems likely that differential expression of the enzyme, especially that of weak or strong intensity, causes some alterations in testicular morphology of homogametic trout. Additionally, the results indicate that an imbalance in sex hormone biosynthesis may provoke the functional alterations in testes of YY males, and, in consequence, negatively affect the fertility of "supermales".