Reproductive performance of lumpfish (Cyclopterus lumpus, L. 1758) females: Effects of integrated photoperiod and temperature manipulations on sexual maturation and spawning.

A successful control of sexual maturation is crucial for year-round production of lumpfish juveniles destined as cleaner fish in Atlantic salmon aquaculture. This study investigated the combined effects of photoperiod and temperature manipulations on sexual maturation and spawning in lumpfish females. Lumpfish juveniles were exposed to simulated natural and nine-month compressed annual photoperiods, with subsequent temperature elevation. Body weight (BW), condition factor (K), gonadosomatic index (GSI), ovarian development, plasma levels of 17ß-estradiol (E2), testosterone (T) and 11-ketotestosterone (11-KT), and spawning were assessed. Compressing the natural photoperiod caused a clear increase and decrease in GSI, T, 11-KT and E2 towards and during the spawning period. Before the temperature elevation, GSI, T, 11-KT, E2 and ovarian development were advanced in the compressed photoperiod. After the temperature elevation, GSI, T, 11-KT and E2 fluctuated more in the compressed photoperiod, while in the natural photoperiod, E2 declined, and GSI, T and 11-KT increased. Spawning was advanced by 1 month in the compressed photoperiod compared to the natural photoperiod. Temperature elevation led to higher levels, earlier peaks and declines of T, 11-KT or E2 in both photoperiods, and advanced spawning by 1.5 months in the compressed photoperiod compared to the natural photoperiod. Temperature elevation also led to increased ovulation recruitment and increased cumulative weight of spawned eggs in the natural photoperiod. Compressing the natural photoperiod and elevating temperature can thus advance sexual maturation and spawning in lumpfish females. Due to the lower amounts of spawned egg weights in the high temperature compressed photoperiod, further studies on effects of photoperiod and timing of temperature manipulations on spawning, fecundity and egg quality could optimize the photothermal manipulations on lumpfish broodstock.

Ultrasonic imaging as a means of monitoring gonadal development in lumpfish (Cyclopterus lumpus).

The commercial farming of juvenile lumpfish requires monitoring of gonadal development to achieve synchronized production. Conventional methods such as gonadosomatic index (GSI), sex hormone analyses, gonadal histology, endoscopy, and gene expression analyses are costly, invasive, and often involve sacrificing the fish. We assessed the efficiency of ultrasound as a non-invasive method for monitoring gonadal development in lumpfish. Based on ultrasound observations, we categorized the fish into six stages; F0 to F5 for females and M0 to M5 for males, that represented maturity levels from immature to spent. Importantly, the ultrasound gonadal stages aligned with histological gonadal stages. Additionally, ultrasound stages aligned with profiles of GSI, testosterone (T), 11-ketotestosterone, and 17ß-estradiol throughout gonadal development including the spawning period. Moreover, these parameters exhibited significant positive correlations with each other reflecting their parallel trends during gonadal development. To minimize the frequency of ultrasound usage and fish handling, we established F3 and M3/M4 as arbitrary thresholds for identifying ripe females and males, respectively. By using these thresholds, the need for regular ultrasound monitoring could be reduced during most of the rearing period. Ultrasound proves to be useful and reliable for monitoring gonadal development in lumpfish, enabling synchronized production of juvenile fish.

Sex steroid dynamics and mRNA transcript profiles of growth- and development-related genes during embryogenesis following induced follicular maturation in European eel.

Hormones and mRNA transcripts of maternal origin deposited in the egg may affect early embryonic development in oviparous species. These hormones include steroids, such as estradiol-17ß (E2), testosterone (T), 11-ketotestosterone (11-kt), 17α,20ß-dihydroxy-4-pregnen-3-one (DHP), and cortisol, which also play an important role in fish reproduction. In European eel, Anguilla anguilla, which does not reproduce naturally in captivity, vitellogenesis in female broodstock is commonly induced by administration of salmon or carp pituitary extract (PE) as an exogenous source of gonadotropins, while follicular maturation is stimulated by a priming dose of PE followed by provision of DHP as a maturation inducing hormone. In this regard, the main purpose of the present study was to evaluate effects of induced follicular maturation on reproductive success in European eel, focusing on maternal transfer and dynamics of steroids and mRNA transcripts of growth- and development-related genes throughout embryogenesis. The results showed that maternal blood plasma concentrations of E2, T and DHP were reflected in the unfertilized eggs. Moreover, a negative relationship between concentrations of E2 and DHP in eggs and embryos and quality parameters measured as fertilization success, cleavage abnormalities, embryonic survival, and hatch success was found. Concomitant mRNA transcript abundance analysis including genes involved in stress response (hsp70, hsp90), somatotropic axis (gh, igf1, igf2a, igf2b), lipid (cpt1a, cpt1b, pigf5) and thyroid metabolism (dio1, dio2, dio3, thrαb, thrßa, thrßb) varied among unfertilized egg batches. For the majority of genes, mRNA abundance increased during the maternal-to-zygotic transition in connection to activation of the transcription of the embryos own genome. mRNA abundance of dio1, cpt1a and cpt1b throughout embryogenesis was related to embryonic developmental competence. Notably, mRNA abundance of dio3 was positively associated with E2 concentrations, while the mRNA abundance of thrαb was negatively related to T concentrations in the unfertilized eggs, which may suggest an interaction between the thyroid and steroid hormone systems. Altogether, maternal plasma concentrations of E2 and DHP were reflected in the eggs, with high concentrations of these steroids in the eggs being negatively associated with embryonic developmental competence. Additionally, high transcript levels of two of the investigated genes (dio1, cpt1b) were positively associated with embryonic developmental competence. This study reveals maternal transfer of steroids and mRNA transcripts to the eggs, which may be significant contributors to the variability in embryonic survival observed in European eel captive reproduction.

Differential expression of gonadotropin and estrogen receptors and oocyte cytology during follicular maturation associated with egg viability in European eel (Anguilla anguilla).

In captivity, oogenesis and ovarian follicle maturation in European eel can be induced experimentally using hormonal therapy. The follicle's ability to respond effectively to the induction of maturation and ovulation, resulting in viable eggs, depends on the oocyte stage at the time of induction. We hypothesized that variation in the expression of key hormone receptors in the ovary and size of oocyte lipid droplets are associated with changes in oocyte stage. Thus, we induced ovarian follicle maturation using a priming dose of fish pituitary extract followed by the administration of a 17α, 20ß-dihydroxy-4-pregnen-3-one (DHP) injection. Females were then strip-spawned, the eggs were fertilized in vitro, incubated and larval survival was recorded at 3 days post hatch (dph). The expression of gonadotropin receptors (fshr, lhcgr1 and lhcgr2) and estrogen receptors (esr1, esr2a, esr2b, gpera and gperb) was quantified and the size of oocyte lipid droplets measured. Larval survival at 3 dph was used to differentiate high- and low-quality egg batches. Results showed significantly higher abundance of lhcgr1 and esr2a at priming for high-quality egg batches whereas fshr and gperb transcripts were significantly higher at DHP injection for low-quality egg batches. Therefore, high levels of lhcgr1 and esr2a may be important for attaining follicular maturational competence, while high fshr and gperb mRNA levels may indicate inadequate maturational competence. Furthermore, lipid droplet size at DHP and in ovulated eggs was significantly smaller in high-quality egg batches than in low-quality, which indicates that droplet size may be a useful marker of follicular maturational stage.

Interactive effects of dietary composition and hormonal treatment on reproductive development of cultured female European eel, Anguilla anguilla.

Farmed female eels were fed two experimental diets with similar proximate composition but different n-3 polyunsaturated fatty acid (PUFA) levels. Both diets had similar levels of arachidonic acid (ARA), while levels of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) in one diet were approximately 4.5 and 2.6 times higher compared to the other diet, respectively. After the feeding period, each diet group was divided into two and each half received one of two hormonal treatments using salmon pituitary extract (SPE) for 13 weeks: i) a constant hormone dose of 18.75mg SPE/kg initial body weight (BW) and ii) a variable hormone dosage that increased from 12.5mg SPE/kg initial BW to 25mg SPE/kg initial BW. Results showed a significant interaction between diets and hormonal treatments on gonadosomatic index (GSI), indicating that the effect of broodstock diets on ovarian development depends on both nutritional status and hormonal regime. Females fed with higher levels of n-3 series PUFAs and stimulated with the constant hormonal treatment reached higher GSIs than those receiving the variable hormonal treatment. However, when females were fed lower levels of n-3 series PUFAs there was no difference in the effect of hormonal treatments on GSI. We also found that, independent of hormonal treatment, the diet with higher levels of n-3 series PUFAs led to the most advanced stages of oocyte development, such as germinal vesicle migration. Concentration of sex steroids (E2, T, and 11-KT) in the plasma did not differ between diets and hormonal treatments, but was significantly correlated with ovarian developmental stage. In conclusion, increasing dietary levels of n-3 PUFAs seemed to promote oocyte growth, leading to a more rapid progression of ovarian development in European eel subjected to hormonal treatment.

Three nuclear and two membrane estrogen receptors in basal teleosts, Anguilla sp.: Identification, evolutionary history and differential expression regulation.

Estrogens interact with classical intracellular nuclear receptors (ESR), and with G-coupled membrane receptors (GPER). In the eel, we identified three nuclear (ESR1, ESR2a, ESR2b) and two membrane (GPERa, GPERb) estrogen receptors. Duplicated ESR2 and GPER were also retrieved in most extant teleosts. Phylogeny and synteny analyses suggest that they result from teleost whole genome duplication (3R). In contrast to conserved 3R-duplicated ESR2 and GPER, one of 3R-duplicated ESR1 has been lost shortly after teleost emergence. Quantitative PCRs revealed that the five receptors are all widely expressed in the eel, but with differential patterns of tissue expression and regulation. ESR1 only is consistently up-regulated in vivo in female eel BPG-liver axis during induced sexual maturation, and also up-regulated in vitro by estradiol in eel hepatocyte primary cultures. This first comparative study of the five teleost estradiol receptors provides bases for future investigations on differential roles that may have contributed to the conservation of multiple estrogen receptors.

Multiple kisspeptin receptors in early osteichthyans provide new insights into the evolution of this receptor family.

Deorphanization of GPR54 receptor a decade ago led to the characterization of the kisspeptin receptor (Kissr) in mammals and the discovery of its major role in the brain control of reproduction. While a single gene encodes for Kissr in eutherian mammals including human, other vertebrates present a variable number of Kissr genes, from none in birds, one or two in teleosts, to three in an amphibian, xenopus. In order to get more insight into the evolution of Kissr gene family, we investigated the presence of Kissr in osteichthyans of key-phylogenetical positions: the coelacanth, a representative of early sarcopterygians, the spotted gar, a non-teleost actinopterygian, and the European eel, a member of an early group of teleosts (elopomorphs). We report the occurrence of three Kissr for the first time in a teleost, the eel. As measured by quantitative RT-PCR, the three eel Kissr were differentially expressed in the brain-pituitary-gonadal axis, and differentially regulated in experimentally matured eels, as compared to prepubertal controls. Subfunctionalisation, as shown by these differences in tissue distribution and regulation, may have represented significant evolutionary constraints for the conservation of multiple Kissr paralogs in this species. Furthermore, we identified four Kissr in both coelacanth and spotted gar genomes, providing the first evidence for the presence of four Kissr in vertebrates. Phylogenetic and syntenic analyses supported the existence of four Kissr paralogs in osteichthyans and allowed to propose a clarified nomenclature of Kissr (Kissr-1 to -4) based on these paralogs. Syntenic analysis suggested that the four Kissr paralogs arose through the two rounds of whole genome duplication (1R and 2R) in early vertebrates, followed by multiple gene loss events in the actinopterygian and sarcopterygian lineages. Due to gene loss there was no impact of the teleost-specific whole genome duplication (3R) on the number of Kissr paralogs in current teleosts.