Recent insights into egg quality and larval vitality of the European eel Anguilla anguilla.

To date, the eel industry still depends on wild-caught juveniles that are grown to marketable size. There is an urgent need to close the eel life cycle in captivity to make aquaculture independent of the natural population. With this artificial reproduction protocol, yolk-sac larvae can be produced but egg quality may be impaired. Low survival rates and high deformity rates are frequently observed during the first week after hatching. Over the past four years, we have conducted studies with the aim to optimize the artificial reproduction protocol, thereby focussing on increasing egg and larval quality. Weekly carp or salmon pituitary extract (PE) treatment was successfully replaced with recombinant gonadotropins (rGTHs) to mature female eels and produce larvae. 17α,20ß-dihydroxy-4-pregnen-3-one (DHP) was replaced with upstream precursor progesterone (P) to induce the endogenous production of DHP by the female eel. DHP and P were found equally potent in inducing oocyte maturation and ovulation. The effects of antibiotics on larval survival and the occurrence of deformities were investigated. Antibiotic treatment increased survival and decreased the occurrence of deformities indicating bacterial infection as an important cause. A deformity determination key for young eel larvae has been developed that provides a framework of reference for larval deformities which will be instrumental with gaining insights on the reasons behind each larval deformity. These improvements of the artificial reproduction protocol and hatchery practices will contribute to the production of robust eel larvae that survive, grow and metamorphose into juveniles that will later be able to reproduce in captivity.

The induction of oocyte maturation and ovulation in the European eel (Anguilla anguilla): in vitro and in vivo comparison of progesterone with 17α,20ß-dihydroxy-4-pregnen-3-one.

Ovulation in European eel is induced by injection of 17α,20ß-dihydroxy-4-pregnen-3-one (DHP) as the maturation-inducing hormone (MIH). Female eels need to ovulate within 18 h after injection to release good quality eggs. Progesterone (P), as an upstream precursor of DHP, may promote endogenous DHP production and improve egg quality. The purpose of this study was therefore to compare treatment of P with DHP on batch level, in vitro, to determine dose-response effects, and in vivo, at a single dose. For the in vitro experiment, ovarian tissue was extracted and placed in culture plates containing hormone-free medium and media supplemented with the treatment: DHP at 1, 10 and 100 ng mL-1, or P at 10, 100 and 1,000 ng mL-1. At the start of incubation, the folliculated oocytes were sampled for histology, microscopy and qPCR. After incubation for 12 and 18 h, the oocytes were sampled for microscopy and qPCR analysis. For the in vivo experiment, females were either injected with DHP or P at a dose of 2 mg kg-1 to assess their effects on ovulation and reproductive success. At the moment of release, eggs were sampled for RNA sequencing to compare effects of DHP and P on the expression of genes involved in egg quality aspects. Remaining eggs were fertilized and larval viability was recorded. Both DHP and P were able to induce GVBD (DHP at 10 and 100 ng mL-1, P at 100 and 1,000 ng mL-1) in vitro. Expression of genes involved in oocyte maturation and ovulation was similar in vitro for both DHP and P treatments. Regarding the in vivo results, RNAseq results reflected similar DHP and P effects on the expression of genes involved in egg quality aspects. Females injected with either DHP or P ovulated, released eggs, and were equally able to produce larvae without any differences in reproductive success. Our results support the conclusion that DHP and P work equally well in vitro and in vivo. P is more attractive to apply as the price is 3,000 times lower than the price of DHP.

Steroid implants for the induction of vitellogenesis in feminized European silver eels (Anguilla anguilla L.).

Assisted propagation of the European eel will lead to a closed production cycle supplying the aquaculture industry with juvenile glass eels. Females require long-term weekly treatment with pituitary extract (PE), which is stressful and causes abnormalities in oogenesis. We tested the effects of 17α-methyltestosterone (17 MT), as potent androgen activating the androgen receptor, and 17ß-estradiol (E2), as an inducer of vitellogenesis, to shorten the duration of PE treatment.Four groups of feminized eels were subjected to a simulated migration and subsequent injection with implants containing 17 MT (17 MT-group), E2 (E2-group) or 17 MT plus E2 (17 MT + E2-group) to test for synergistic effects, or without any steroids as controls (C-group). The effects of a 2-months treatment were investigated by determining the eye index (EI), hepatosomatic and gonadosomatic index (HSI and GSI, respectively), plasma steroid concentrations by liquid chromatography mass spectrometry (LCMS), gonadal histology, expression of androgen receptors a and b (ara, arb); estrogen receptor 1 (esr1); FSH receptor (fshr); vitellogenin receptor (vtgr) and aromatase (cyp19), and the required number of weekly PE injections to fully mature. For many parameters, both the 17 MT and E2 groups showed an increase vs. controls, with the 17 MT + E2 group showing a synergistic effect, as seen for EI, GSI (3.4 for 17 MT and for E2, 6.6 for 17 MT + E2), oocyte diameter and ara, arb and esr1 expression. Concentrations of almost all focal steroids decreased with simulated migration and steroid treatment. Only eels of the 17 MT-group showed increased expression of cyp19 and of fshr, while fshr expression increased 44-fold in the 17 MT + E2 group, highlighting that co-implantation is most effective in raising fshr mRNA levels. Specific for eels of the E2 groups were vitellogenesis-associated changes such as an increase of HSI, plasma E2, and presence of yolk in the oocytes. Steroid treatments reduced the duration of PE treatment, again synergistically for co-implantation. In conclusion, E2 is necessary to start vitellogenesis, but 17 MT has specific effects on cyp19 and fshr expression. The combination is necessary for synergistic effects and as such, steroid implants could be applied in assisted reproduction protocols for European eel to improve oocyte quality leading to the production of more vital larvae.

What Goes Wrong during Early Development of Artificially Reproduced European Eel Anguilla anguilla? Clues from the Larval Transcriptome and Gene Expression Patterns.

In eels, large variations in larval mortality exist, which would impede the viable production of juvenile glass eels in captivity. The transcriptome of European eel larvae was investigated to identify physiological pathways and genes that show differential regulation between non-viable vs. viable larvae. Expression of genes involved in inflammation and host protection was higher, suggesting that non-viable larvae suffered from microbial infection. Expression of genes involved in osmoregulation was also higher, implying that non-viable larvae tried to maintain homeostasis by strong osmoregulatory adaptation. Expression of genes involved in myogenesis, neural, and sensory development was reduced in the non-viable larvae. Expression of the major histocompatibility complex class-I (mhc1) gene, M-protein (myom2), the dopamine 2B receptor (d2br), the melatonin receptor (mtr1), and heat-shock protein beta-1 (hspb1) showed strong differential regulation and was therefore studied in 1, 8, and 15 days post-hatch (dph) larvae by RT-PCR to comprehend the roles of these genes during ontogeny. Expression patterning of these genes indicated the start of active swimming (8 dph) and feed searching behavior (15 dph) and confirmed immunocompetence immediately after hatching. This study revealed useful insights for improving larval survival by microbial control and salinity reduction.

Energy efficiency of digestible protein, fat and carbohydrate utilisation for growth in rainbow trout and Nile tilapia.

Currently, energy evaluation of fish feeds is performed on a digestible energy basis. In contrast to net energy (NE) evaluation systems, digestible energy evaluation systems do not differentiate between the different types of digested nutrients regarding their potential for growth. The aim was to develop an NE evaluation for fish by estimating the energy efficiency of digestible nutrients (protein, fat and carbohydrates) and to assess whether these efficiencies differed between Nile tilapia and rainbow trout. Two data sets were constructed. The tilapia and rainbow data set contained, respectively, eight and nine experiments in which the digestibility of protein, fat and energy and the complete energy balances for twenty-three and forty-five diets was measured. The digestible protein (dCP), digestible fat (dFat) and digestible carbohydrate intakes (dCarb) were calculated. By multiple regression analysis, retained energy (RE) was related to dCP, dFat and dCarb. In tilapia, all digestible nutrients were linearly related to RE (P<0·001). In trout, RE was quadratically related to dCarb (P<0·01) and linearly to dCP and dFat (P<0·001). The NE formula was NE=11·5×dCP+35·8×dFAT+11·3×dCarb for tilapia and NE=13·5×dCP+33·0×dFAT+34·0×dCarb-3·64×(dCarb)2 for trout (NE in kJ/(kg0·8×d); dCP, dFat and dCarb in g/(kg0·8×d)). In tilapia, the energetic efficiency of dCP, dFat and dCarb was 49, 91 and 66 %, respectively, showing large similarity with pigs. Tilapia and trout had similar energy efficiencies of dCP (49 v. 57 %) and dFat (91 v. 84 %), but differed regarding dCarb.

Ultrasonography to assist with timing of spawning in European eel.

Ovulation in the European eel can be induced by injection of DHP (17α,20ß-dihydroxy-4-pregnen-3-one). The timing of injection is based on the developmental stage of the oocytes. The oocyte stage is determined by invasive biopsy or through external indicators of the oocyte hydration response: body weight index (BWI) and body girth index (BGI). However, in European eel, BWI and BGI are inaccurate indicators because the individual hydration response is highly variable. The aim of this study was to identify indicators of oocyte maturation non-invasively by applying ultrasonography. Ultrasound parameters that were determined were the body wall thickness; diameter of the cloaca opening; grey scale median (GSM) of the ovary, and diameters (vertical and horizontal) and surface area of ovary, liver and swim bladder. Data of farmed and wild eels were combined for correlation analyses and principal component analyses (PCA). Ovary and liver parameters were not correlated with the average oocyte stage. Body wall thickness was negatively correlated to the average oocyte stage but variability was high. The swim bladder vertical diameter (Sbv) was negatively correlated with the average oocyte stage and with the oocyte diameter. Swim bladder area (Sba) was also negatively correlated to the average oocyte stage but less accurate. Results showed that during the final stages of oocyte maturation, the swim bladder becomes smaller. This is most probably due to the pressure that is caused by the increasing ovaries as a result of the oocyte hydration response. Females with an average oocyte stage between 4.5 and 7.0 and an average oocyte diameter higher than 800 µm, had Sbv values decreasing from 4.3 to 2.4 mm and Sba values from 52 down to 11 mm2. The correlation between Sbv and oocyte stage is more strict than for BWI or BGI which makes Sbv not only a parameter that can be determined non-invasively but also one that is more accurate in indicating the oocyte developmental stage. Therefore, ultrasonography, and in particular the ultrasound imaging of the swim bladder, represents a useful tool to assist with the timing of spawning as induced by injection of DHP.

Isoenergetic replacement of fat by starch in diets for African catfish (Clarias gariepinus): effect on water fluxes in the gastro intestinal tract.

The effect of an isoenergetic replacement of dietary fat by starch, on chyme characteristics and water fluxes in the gastro intestinal tract (GIT) was assessed. Adult African catfish (Clarias gariepinus) were fed a starch (SD) or fat (FD) diet and groups of fish were dissected at 2, 5 and 8 h after the consumption of a single meal. Chyme was collected quantitatively and was analysed for osmolality and dry matter (DM) content. Postprandial water fluxes were calculated, while using yttrium oxide (Y(2)O(3)) as an inert marker to account for the absorption of DM along the GIT. The largest differences in chyme characteristics between diets were observed in the stomach and decreased towards subsequent compartments. A high initial osmotic pressure was measured in the stomach for both diets (up to 498 ± 2 mOsm kg(-1)) and was likely the driver for the endogeneous water influx to this compartment. Large additions of water were recorded to the stomach and proximal intestine for both diets and absorption of water took place in the mid- and distal intestine. Interestingly, the dietary treatment had an impact on water balance in the stomach and proximal intestine of the fish, but not in the mid- and distal intestine. A strong complementary relationship suggested that 59% of the water fluxes in the proximal intestine could be explained by previous additions to the stomach. Therefore, a higher dietary inclusion of starch led to a shift in water additions from the proximal intestine to the stomach. However, the sum of water additions to the GIT was not different between diets and was on average 6.52 ± 0.85 ml water g(-1) DM. The interactions between osmoregulation and digestion, in the GIT of fed freshwater fish, deserve further attention in future research.