Effects of Stocking Density and Pre-Slaughter Handling on the Fillet Quality of Largemouth Bass (Micropterus salmoides): Implications for Fish Welfare.

There is currently insufficient acknowledgment of the relationship between fish welfare and ultimate fillet quality. The purpose of this study was to assess the impacts of pre-slaughter handling and stocking density as fish welfare markers on fillet quality of largemouth bass (Micropterus salmoides). Fish from three stocking densities of 35, 50, and 65 kg·m-3 were reared in a recirculating aquaculture system (RAS) for 12 weeks and received commercial feed. Ultimately, the fish were either stunned with percussion on the head (control group) or subjected to air exposure for 3 min (anoxia group) before stunning and subsequent collection of blood and fillet samples. Western blot analysis revealed the degradation of actin in both groups. Additionally, higher oxidation progress and lower hardness and pH were observed in anoxia compared to the control group. We observed higher hardness at 35 kg·m-3 in anoxia compared to 50 and 65 km-3. The initial hardness values at 35, 50, and 65 km-3 were 1073, 841, and 813 (g) respectively in the anoxia group. Furthermore, the anoxia and control groups had rigor mortis after 6 and 10 h, respectively. Cortisol and glucose levels, and oxidative enzymes activity were higher in anoxia than in the control group. In conclusion, oxidation induced by anoxia likely plays a crucial role as a promoter of the quality deterioration of largemouth bass fillets.

In vitro post-ovulatory oocyte ageing in grass carp Ctenopharyngodon idella affects H4K12 acetylation pattern and histone acetyltransferase activity.

Delayed fertilization leads to the ageing of post-ovulatory oocytes and reduces the developmental competence of arising embryos. Little information is available about the molecular processes during fish oocyte ageing. The current study investigated the functional consequences of oocyte ageing in grass carp Ctenopharyngodon idella embryos. In addition, the dynamics of selected post-transcriptionally modified histones (acetylation of H3K9, H3K14, H4K5, H4K8, H4K12, and H4K16) were analyzed during oocyte ageing. Ovulated oocytes were aged in vitro for 4 h in the laboratory incubator at 20 °C and studied for selected post-translational modification of histones. In addition, histone acetyltransferase activity was investigated as an important regulator of histone acetylation modification. The results indicated a significant decrease in oocyte fertilizing ability through 1 h of post-ovulatory ageing, and a complete loss of egg fertilizing abilities was detected at 4-h aged oocytes. Furthermore, post-ovulatory oocyte ageing for 1 and 4 h led to decreased levels of H4K12 acetylation. The activity of histone acetyltransferases increased significantly after ageing of the oocytes for 30 h in vitro. This modification may partly contribute to explaining the failures of egg viability and embryo development in the offspring from the aged oocytes. The results are the first to report histone modifications as a crucial epigenetic regulator during oocyte ageing in fish and might also benefit other vertebrates.

Fine ultrastructural features of germ cells and spermatozoa during spermatogenesis in the European grayling, Thymallus thymallus (Teleostei, Salmoniformes, Salmonidae).

This study aimed to examine the ultrastructure of spermatogenic stages and mature spermatozoa in the European grayling, Thymallus thymallus. The testes were examined microscopically with a transmission electron microscope to find out details of the structure and morphology of the grayling germ cells, spermatozoa and some somatic cells. The grayling testis has a tubular shape, with cysts or clusters of germ cells within seminiferous lobules. The spermatogenic cells, including spermatogonia, spermatocytes, and spermatids, can be found along seminiferous tubules. There are electron-dense bodies in germ cells from the primary spermatogonia to secondary spermatocyte stages. These undergo mitosis to reach the secondary spermatogonia stage, when they form primary and secondary spermatocytes. Spermatids undergo three different stages of differentiation during spermiogenesis, characterized by the level of chromatin condensation, elimination of cytoplasm, and the occurrence of the flagellum. The midpiece of spermatozoa is short and contains spherical or ovoid mitochondria. The sperm flagellum has an axoneme with nine doublets of peripheral microtubules and two central microtubules. The result of this study is valuable to be used as a standard reference for germ cell development, which is of great importance to get a clear insight into the process of grayling breeding practice.

Defatted black soldier fly (Hermetia illucens) in pikeperch (Sander lucioperca) diets: Effects on growth performance, nutrient digestibility, fillet quality, economic and environmental sustainability.

The use of insect meal in aquafeed formulations has recently gained attention. Detailed knowledge about the inclusion levels for pikeperch (Sander lucioperca), a promising candidate for intensive aquaculture in Europe remains, however, fragmented. In the present study, 4 isoproteic (45% dry matter) and isoenergetic (21 MJ/kg) diets were formulated, including a control diet (H0) containing 30% fishmeal (FM) on an as-fed basis and the other 3 diets in which FM protein was replaced by defatted black soldier fly (Hemetia illucens) meal (HIM) at 25%, 50%, and 100% (diet abbreviation H9, H18 and H36, corresponding to an inclusion level of 9%, 18% and 36%, respectively). The feeding trial was performed in triplicate groups of 50 juvenile pikeperch (mean weight, 68.7 g) fed with experimental diets for 84 d during which the growth performance, nutrient digestibility, fillet quality and economic and environmental sustainability of rearing pikeperch were evaluated. Our findings indicated that pikeperch in H0, H9, and H18 groups displayed better results regarding growth performance indices, except for survival rate where no significant difference among groups was recorded (P = 0.642). A significantly lower organ-somatic index, including hepatosomatic, viscerosomatic and perivisceral fat index, was found in fish in H18 groups than other groups (P < 0.05). Inclusion of HIM affected the digestibility of the nutrients and resulted in an almost linear reduction in the apparent digestibility coefficient of dry matter and protein. Concerning the fillet quality, dietary HIM negatively affected the protein and ash contents of the fish fillets, while the crude fat remained unchanged. Dietary HIM did not significantly modify total saturated, monounsaturated and polyunsaturated fatty acids in the fillets of fed pikeperch (P > 0.05) but did reduce total n-3 fatty acids (P = 0.001) and increased total n-6 (P < 0.001). Increasing inclusion levels of HIM reduced the environmental impacts associated with fish in-to-fish out ratio but entailed heavy burdens on energy use and eutrophication. Low and moderate inclusion levels of HIM did not negatively affect land use and water use compared to an HIM-free diet (P > 0.05). The addition of HIM at a level as low as 9% elicited a similar carbon footprint to that of the control diet. The economic conversion ratio and economic profit index were negatively affected at increased insect meal inclusion levels. This study has shown that the incorporation of HIM in feed formulations for pikeperch is feasible at inclusion levels of 18% without adverse effects on growth performance parameters. The feasibility also highlighted the environmental benefits associated with land use and marine resources required to produce farmed fish.

Considering Two Aspects of Fish Welfare on African Catfish (Clarias gariepinus) Fillet throughout Postmortem Condition: Efficiency and Mechanisms.

Knowledge about fish welfare and its impact on fish fillet quality is still insufficient. Therefore, the influence of two aspects of fish welfare (slaughtering method: bled and unbled fish; fish stock densities: 90, 120, and 150 kg·m-3) on African catfish fillet quality during postmortem conditions was investigated. The aim of study was to determine (i) the efficiency of bleeding on oxidation progress and (ii) the influence of stock density on fillet quality. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) showed a higher protein loss in the unbled than in the bled groups, especially in the heavy myosin chain (MHC) band. However, density did not show any influence on protein profile. Western blot analysis showed fewer oxidized carbonyls in the bled than in the unbled groups; higher oxidation development, microbial growth, and lower hardness were observed in unbled fillets. Additionally, hardness was higher at 90 and 120 kg·m-3 densities in bled fillet compared to 150 kg·m-3. The first three days of storage showed a higher oxidation rate in unbled fillets than in bled fillets, confirming the contribution of hemoglobin to oxidation development with different mechanisms of protein oxidation. The obtained results revealed the same fillet quality in all aspects at either 90 or 120 (kg·m-3) stock densities, which would suggest 120 kg·m-3 for the fishery industry. However, higher stocking density in this study would not be appropriate for fish welfare.

Spawning Performance and Sex Steroid Levels in Female Pikeperch Sander lucioperca Treated with Poly(lactic-co-glycolic acid) Microparticles.

Pikeperch Sander lucioperca is a piscivorous species considered a promising candidate for the diversification of intensive aquaculture. This study aimed to determine the effect of a sustained-release delivery system incorporating mammalian gonadotropin-releasing hormone agonist (mGnRHa) into poly(lactic-co-glycolic acid) (PLGA) microparticles on the sex steroid levels and aspects of artificial reproduction of pikeperch. Fish were divided into four groups and injected with 20 µg mGnRHa/kg, 5-day release microparticles encapsulated with 5 µg GnRHa/kg BW (PLGA 5), 20 µg GnRHa/kg (PLGA 20), or 1 mL/kg 0.9% NaCl (control). Cumulative percentage ovulation was 100% in the PLGA 5 group, significantly higher than in other tested groups. No differences among groups were observed in latency or fecundity. The level of 11-ketotestosterone (11-KT) peaked at 40 h post-injection, and was sustained during ovulation, in all treated groups. The 17ß-estradiol (E2) concentration increased in the mGnRHa-only group immediately after hormone injection, while both PLGA groups showed a reduction in E2 after injection, continuing to decrease until ovulation. A low dose of mGnRHa in PLGA microparticles significantly improves induction of ovulation and results in acceptable reproductive performance, which may positively affect pikeperch production under controlled conditions.

Bioenergetic Pathways in the Sperm of an Under-Ice Spawning Fish, Burbot (Lota lota): The Role of Mitochondrial Respiration in a Varying Thermal Environment.

Regarding the sperm of cold-water fish, the contributions of different bioenergetic pathways, including mitochondrial respiration, to energy production at the spawning temperature and its adaptation at the maximum critical temperature (CTmax) are unclear. The roles of glycolysis, fatty acid oxidation, oxidative phosphorylation (OXPHOS) at 4 °C, and OXPHOS at 15 °C for energy production in burbot (Lota lota) spermatozoa were studied by motility and the oxygen consumption rate (OCR) (with and without pathway inhibitors and the OXPHOS uncoupler). At both temperatures, the effects of the inhibitors and the uncoupler on the motility duration, curvilinear velocity, and track linearity were insignificant; in addition, the OCRs in activation and non-activation media differed insignificantly and were not enhanced after uncoupler treatment. After inhibitor treatment in both media, OXPHOS was insignificantly different at the 2, 30, and 60 s time points at 4 °C but was reduced significantly at the 30 and 60 s time points after treatment with sodium azide at 15 °C. In conclusion, for burbot sperm at both the spawning temperature and the CTmax, the energy synthesized via OXPHOS during motility was insufficient. Therefore, the majority of the energy required to sustain motility was derived from pre-accumulated energy produced and stored during the quiescent state of the spermatozoa.

Histone Acetylation Dynamics during In Vivo and In Vitro Oocyte Aging in Common Carp Cyprinus carpio.

Aging is the most critical factor that influences the quality of post-ovulatory oocytes. Age-related molecular pathways remain poorly understood in fish oocytes. In this study, we examined the effect of oocyte aging on specific histone acetylation in common carp Cyprinus carpio. The capacity to progress to the larval stage in oocytes that were aged for 28 h in vivo and in vitro was evaluated. Global histone modifications and specific histone acetylation (H3K9ac, H3K14ac, H4K5ac, H4K8ac, H4K12ac, and H4K16ac) were investigated during oocyte aging. Furthermore, the activity of histone acetyltransferase (HAT) was assessed in fresh and aged oocytes. Global histone modifications did not exhibit significant alterations during 8 h of oocyte aging. Among the selected modifications, H4K12ac increased significantly at 28 h post-stripping (HPS). Although not significantly different, HAT activity exhibited an upward trend during oocyte aging. Results of our current study indicate that aging of common carp oocytes for 12 h results in complete loss of egg viability rates without any consequence in global and specific histone modifications. However, aging oocytes for 28 h led to increased H4K12ac. Thus, histone acetylation modification as a crucial epigenetic mediator may be associated with age-related defects, particularly in oocytes of a more advanced age.

Oocyte Ageing in Zebrafish Danio rerio (Hamilton, 1822) and Its Consequence on the Viability and Ploidy Anomalies in the Progeny.

Fish egg quality can be markedly influenced by the oocyte age after ovulation. In this study, we examined the duration of oocyte ageing in the zebrafish (Danio rerio) and whether prolonged ageing is associated with the incidence of ploidy anomalies in the resulting embryos. Oocytes were incubated in vitro for 6 h post-stripping (HPS) at 26 °C and fertilized at 2-h intervals. Meanwhile, for eggs fertilized immediately after stripping, the fertilization, embryo survival, and hatching rates started at ~80%; these rates decreased to 39%, 24%, and 16%, respectively, for oocytes that had been stored for 4 h (p ˂ 0.05), and there was an almost complete loss of egg viability at 6 HPS. Furthermore, almost 90% of the embryos derived from 6-h aged oocytes died prior to hatching, and all larvae originating from 4- and 6-h aged oocytes showed malformations. The proportion of ploidy abnormal embryos was significantly greater at 4 HPS (18.5%) than at either 0 or 2 HPS (4.7% and 8.8%, respectively). The results revealed that zebrafish oocytes retained their fertilization potential for up to 2 h after stripping at 26 °C and indicated the contribution of post-ovulatory oocyte ageing in the occurrence of ploidy anomalies in the resulting embryos.

Natural Feed Additives Modulate Immunity and Mitigate Infection with Sphaerospora molnari (Myxozoa:Cnidaria) in Common Carp: A Pilot Study.

Myxozoans are a diverse group of cnidarian parasites, including important pathogens in different aquaculture species, without effective legalized treatments for fish destined for human consumption. We tested the effect of natural feed additives on immune parameters of common carp and in the course of a controlled laboratory infection with the myxozoan Sphaerospora molnari. Carp were fed a base diet enriched with 0.5% curcumin or 0.12% of a multi-strain yeast fraction, before intraperitoneal injection with blood stages of S. molnari. We demonstrate the impact of these treatments on respiratory burst, phagocytosis, nitric oxide production, adaptive IgM+ B cell responses, S. molnari-specific antibody titers, and on parasite numbers. Both experimental diets enriched B cell populations prior to infection and postponed initial parasite proliferation in the blood. Curcumin-fed fish showed a decrease in reactive oxygen species, nitric oxide production and B cell density at late-stage infection, likely due to its anti-inflammatory properties, favoring parasite propagation. In contrast, multi-strain yeast fraction (MsYF)-fed fish harbored the highest S. molnari-specific antibody titer, in combination with the overall lowest parasite numbers. The results demonstrate that yeast products can be highly beneficial for the outcome of myxozoan infections and could be used as effective feed additives in aquaculture.

First identification of dopamine receptors in pikeperch, Sander lucioperca, during the pre-ovulatory period.

Dopamine (DA) is a ubiquitous neurotransmitter exerting a range of pleiotropic actions through two DA receptor families, the D1 and the D2. To date in vertebrates, a maximum of four receptor subtypes have been identified within the D1 family, D1 (former D1A), D5 (former D1B), D6 (former D1C and D1D) and D7 (former D1E), while the D2 family encloses five subtypes, D2, D3, D4, D8 (former D2like or D2l) and D9 (former D4-related sequence or D4-rs). In teleosts, no study has investigated in parallel all the DA receptors to identify and localize the whole receptor repertoire from both families. In pikeperch, Sander lucioperca, a species of interest for aquaculture development, the existence, number and location of the DA receptors are totally unknown. To address these questions, RNA-seq with de novo transcriptome reconstruction, functional annotation and phylogenetic analysis were performed to characterize the transcript repertoire of DA receptors in the brain of female pikeperch at the pre-ovulatory period. Ten different cDNA were identified and showed to belong to the D1 family: two D1, one D5a, one D6a and one D6b and to the D2 family: two spliced variants of D2, one D3, one D8 and one D9. Unlike zebrafish, the subtypes D4 and D7 have not yet been isolated in pikeperch. As expected D1, D3, D8 and D9 are mostly expressed in brain parts except for the cerebellum (D1 and D3). The inter-species differences in the number of DA receptors and the inter-organ differences in the gene expression of all receptors support the complexity of the dopaminergic actions in vertebrate.

Improvements on Live Feed Enrichments for Pikeperch (Sander lucioperca) Larval Culture.

This study focused on supplementing pikeperch (Sander lucioperca) larvae with rotifers fed with Chlorella vulgaris during the first 15 days post hatching (dph). Larvae were fed a combination of rotifers and artemia under three different enrichments: A) Nannochloropsis occulata, B) Chlorella vulgaris, and C) a commercial enrichment-Selco, Spresso from INVE. After 17 days from the trial initiation differences were found between treatments on survival rate, myomere height (MH), fatty acid composition, and stress tolerance. In terms of survival, larvae from treatment b (74.5%) and c (66%) excelled over the control (a) treatment (59%). Furthermore, larvae from both the Chlorella (b) and the Selco (c) treatments showed more resilience to stress conditions (10% and 37% reduction in mortality) when exposed to high salinity conditions (18ppt) for 3 h (stress response). Overall, larvae from treatments b and c performed better than those receiving a non-enriched diet (a), likely due to the higher levels of Essential Fatty Acids (EFA) and the ability of pikeperch to desaturate and elongate fatty acids (FA) with 18 carbons to LC PUFAs (Polyunsaturated Fatty Acids). The present study provides valuable input for designing improved feeding protocols, which will increase the efficiency of pikeperch larval culture.

Molecular Characterization of PGC-1ß (PPAR Gamma Coactivator 1ß) and its Roles in Mitochondrial Biogenesis in Blunt Snout Bream (Megalobrama amblycephala).

This study aimed at achieving the molecular characterization of peroxisome proliferator-activated receptor-gamma coactivator 1ß (PGC-1ß) and exploring its modulatory roles in mitochondria biogenesis in blunt snout bream (Megalobrama amblycephala). A full-length cDNA of PGC-1ß was cloned from liver which covered 3110 bp encoding 859 amino acids. The conserved motifs of PGC-1ß family proteins were gained by MEME software, and the phylogenetic analyses showed motif loss and rearrangement of PGC-1ß in fish. The function of PGC-1ß was evaluated through overexpression and knockdown of PGC-1ß in primary hepatocytes of blunt snout bream. We observed overexpression of PGC-1ß along with enhanced mitochondrial transcription factor A (TFAM) expression and mtDNA copies in hepatocytes, and its knockdown led to slightly reduced NRF1 expression. However, knockdown of PGC-1ß did not significantly influence TFAM expression or mtDNA copies. The alterations in mitochondria biogenesis were assessed following high-fat intake, and the results showed that it induces downregulation of PGC-1ß. Furthermore, significant decreases in mitochondrial respiratory chain activities and mitochondria biogenesis were observed by high-fat intake. Our findings demonstrated that overexpression of PGC-1ß induces the enhancement of TFAM expression and mtDNA amount but not NRF-1. Therefore, it could be concluded that PGC-1ß is involved in mitochondrial biogenesis in blunt snout bream but not through PGC-1ß/NRF-1 pathway.

Use of Lactic Acid Bacteria During Pikeperch (Sander lucioperca) Larval Rearing.

This trial tested the use of lactic acid bacteria (LAB) on pikeperch (Sander lucioperca) larvae during their first feeding. The trial included the use of two probiotic treatments and one control (no probiotics). Pikeperch larvae were exposed to LAB as follows: (1) the live feed (Treatment 1, live feed) or (2) via the live feed and the larval rearing water (Treatment 2, probiotic). Significant differences were found between the treatments in terms of total length (TL), myomere height (MH), overall survival, and the tolerance to a high salinity challenge. Larvae exposed to LAB via both the live feed and the rearing water had a significantly higher overall survival rate (85%) than the other two treatments at 21 dph. When both treatments were subjected to high salinity rates (18 parts per thousand (ppt)), both treatments exposed to LAB demonstrated higher survival rates than the control treatment (28% and 40% survival rate at 180 min for the live feed and probiotic treatments, respectively, as compared with a 100% mortality rate at 150 min for the control). At the same time, larvae exposed to the probiotic treatment had a significantly higher TL as compared to the control after 12 and 21 days post hatch (dph) (probiotic 7.13 ± 0.21 and 11.71 ± 1.1 mm, control 5.86 and 10.79 mm at 12 and 21 dph, respectively). The results suggest that the use of LAB in both the live feed and the rearing water has a positive effect on pikeperch larval quality by strengthening their resilience to stress conditions, as well as improving the growth and survival rates.

Silver carp (Hypophthalmichthys molitrix) can non-mechanically digest cyanobacteria.

The growth of cyanobacteria (blue-green algae) is a typical phenomenon in water bodies worldwide. The use of silver carp (Hypophthalmichthys molitrix) to reduce excessive phytoplankton development is controversial. In the case of cyanobacteria, many of which are toxic, understanding their possible digestion mechanism by fish is particularly desirable. A unique methodical approach, which consists of applying intestinal contents or extracts to a cyanobacteria culture, was used. Unicellular cyanobacteria (Cyanothece) were incubated in vitro with bile, contents of different parts of the intestinal tract, and cytosolic and microsomal extracts of the intestinal tissue of silver carp. The abundance of cyanobacteria decreased in all treatments containing either exclusively bile or its combination with intestinal contents. This research provides the first evidence of non-mechanical digestion of cyanobacteria by silver carp. Cyanobacteria incubated with intestinal contents or extracts reached mostly higher abundances than those incubated with the nutrient medium. The existence of non-mechanical digestion mediated via intestinal contents and extracts or its compensation connected with organic substance uptake is discussed.

Fin damage and growth parameters relative to stocking density and feeding method in intensively cultured European perch (Perca fluviatilis L.).

We evaluated the relationship of stocking density to survival, growth performance and fin condition of European perch Perca fluviatilis with hand feeding and self-feeders. Hand-fed perch (body weight 19.1 ± 5.1 g and total length 107 ± 9 mm) were reared at 0.5, 1.0, 1.5 and 2.0 fish/L. Self-feeding perch (body weight 25.4 ± 3.9 g and total length 128 ± 7 mm) were reared at stocking densities of 0.6, 1.0 and 1.4 fish/L. Pond-reared perch served as a comparison group for fin damage assessment. We found no differences in survival rate among stocking densities with either feeding method. Hand-fed fish displayed the highest weight gain and SGR at stocking density of 0.5 fish/L. The self-feeding fish showed a non-linear association of weight gain with stocking density with the highest growth at 1.0 fish/L. Fin length was noticeably greater in pond-reared fish compared with RAS-reared fish regardless of feeding method. In both experiments, fin length relative to standard length showed a negative relationship with stocking density, with pectoral fins showing the greatest effect. Fin condition deteriorated with increasing stocking density, and growth was highest at 0.5 and 1.0 fish/L in hand-fed and self-feeding fish, respectively.

Ultrastructural feature of spermatogenic cells and spermatozoon in cultured burbot Lota lota.

Testis development and ultrastructure of spermatogenic cells and spermatozoa of burbot Lota lota, a commercially important cold freshwater fish, were studied by light and transmission electron microscopy. Spermatogonia, spermatocytes, spermatids, and spermatozoa are distributed along the seminiferous tubules. Electron-dense bodies appear in germ cells from primary spermatogonia to secondary spermatocytes. We identified three distinct stages of spermatid cell differentiation based on chromatin condensation, development of the flagellum, formation of a nuclear fossa, and elimination of excess cytoplasm. Spermatozoa were anacrosomal and characterized by location of the centrioles outside the nuclear fossa and incomplete perpendicular arrangement of the centrioles. The sperm flagellum displayed an axoneme with nine doublets of peripheral microtubules and two central microtubules. These results provide valuable information for burbot taxonomy and may clarify the process of spermatogenesis for this species.

Effects of temperature on sperm motility of burbot Lota lota: spontaneous activation and calcium dependency.

Several factors regulating activation of spermatozoon motility in Eurasian burbot, Lota lota, including osmolality, calcium (Ca2+ ) ions, and temperature were investigated. Spermatozoon motility in Eurasian burbot, Lota lota was assessed at 4 and 30°C in seminal fluid, isotonic media (with and without Ca2+ ) and hypotonic media (with and without Ca2+ ). Spermatozoa were spontaneously activated in seminal fluid at 20°C and the maximum motility was recorded at 30°C, which is out of the spawning temperature range, indicating that no risk of activation occurs during routine semen handling in artificial insemination. Initiation of spermatozoon motility in L. lota is mediated by Ca2+ and sensitivity to Ca2+ is dependent on temperature.

The possible involvement of oxidative stress in the oocyte ageing process in goldfish Carassius auratus (Linnaeus, 1758).

Decreasing egg quality following oocyte ageing is a major restricting factor for the breeding programs. The mechanisms behind this process has not yet been clarified. To examine the possible involvement of oxidative stress in the oocyte ageing process, the relative mRNA abundance of specific transcripts were determined in oocytes collected from 6 females and incubated in vitro for 18 hours post stripping at 20 °C in goldfish Carassius auratus. During the 18 hour-post-stripping ageing of the oocytes, relative mRNA levels of candidate transcripts involved in oxidative injury, mitochondrial function and stress response, cell cycles, apoptosis, reproduction and germ line speciation and developmental competence were measured by real-time PCR. None of the relative mRNA abundance of the examined genes were significantly altered through oocyte ageing. In addition, the amount of thiobarbituric acid reactive substances (TBARS), an indicator of lipid peroxidation, did not change over time following stripping. The activity of the antioxidant enzymes also remained constant during oocyte ageing. The results of the current study indicated that oxidative stress unlikely plays a role as an initiator or promotor in the progress of oocyte ageing in goldfish.

The kinetics of cellular and humoral immune responses of common carp to presporogonic development of the myxozoan Sphaerospora molnari.

BACKGROUND: Sphaerospora molnari is a myxozoan parasite causing skin and gill sphaerosporosis in common carp (Cyprinus carpio) in central Europe. For most myxozoans, little is known about the early development and the expansion of the infection in the fish host, prior to spore formation. A major reason for this lack of information is the absence of laboratory model organisms, whose life-cycle stages are available throughout the year. RESULTS: We have established a laboratory infection model for early proliferative stages of myxozoans, based on separation and intraperitoneal injection of motile and dividing S. molnari stages isolated from the blood of carp. In the present study we characterize the kinetics of the presporogonic development of S. molnari, while analyzing cellular host responses, cytokine and systemic immunoglobulin expression, over a 63-day period. Our study shows activation of innate immune responses followed by B cell-mediated immune responses. We observed rapid parasite efflux from the peritoneal cavity (< 40 hours), an initial covert infection period with a moderate proinflammatory response for about 1-2 weeks, followed by a period of parasite multiplication in the blood which peaked at 28 days post-infection (dpi) and was associated with a massive lymphocyte response. Our data further revealed a switch to a massive anti-inflammatory response (up to 1456-fold expression of il-10), a strong increase in the expression of IgM transcripts and increased number of IgM+ B lymphocytes, which produce specific antibodies for the elimination of most of the parasites from the fish at 35 dpi. However, despite the presence of these antibodies, S. molnari invades the liver 42 dpi, where an increase in parasite cell number and indistinguishable outer cell membranes are indicative of effective exploitation and disguise mechanisms. From 49 dpi onwards, the acute infection changes to a chronic one, with low parasite numbers remaining in the fish. CONCLUSIONS: To our knowledge, this is the first time myxozoan early development and immune modulation mechanisms have been analyzed along with innate and adaptive immune responses of its fish host, in a controlled laboratory system. Our study adds important information on host-parasite interaction and co-evolutionary adaptation of early metazoans (Cnidaria) with basic vertebrate (fish) immune systems and the evolution of host adaptation and parasite immune evasion strategies.