Sperm contamination by urine in Senegalese sole (Solea senegalensis) and the use of extender solutions for short-term chilled storage

Methods are needed to manage the sperm of Senegalese sole (Solea senegalensis), which will enable the industry to use artificial fertilisation to reproduce hatchery raised sole and implement breeding programs. The present study aimed to (a) describe the male reproductive and urinary system, (b) describe the effects of urine contamination on sperm quality and (c) examine the use of extenders for short term chilled storage of sole sperm. Nine males were dissected to describe the male reproductive and urinary system. A total of 49 males were examined and 32 (65.3%) provided adequate sperm samples of the study. Initially the samples were described by appearance (colour, transparency and fluidity) and sub-samples analysed for sperm quality, urea concentration, osmolality, pH and protein concentration. Cell concentration and sperm quality parameters, percentage motility, curvilinear velocity (VCL) and average path velocity (VAP), were measured using ImageJ CASA. Control samples and samples diluted (1:3) in six different extender solutions (modified Leibovitz, Ringer, NAM, Sucrose, Stor Fish® and Marine Freeze®) were stored short-term (4 °C) and tested zero, three, six and 24 h after collection. The close proximity of the reproductive and the urinary systems, especially the sperm ducts being attached to the urinary bladder makes obtaining sperm without urine contamination appear difficult. All the samples appeared to be contaminated with urine. Samples that appeared to be contaminated with urine (yellow colour) had similar spermatozoa cell concentration and urea concentration as samples that appeared not to be contaminated with urine (whitish colour), although motility was significantly lower in yellow samples. Seminal plasma urea concentration was positively correlated with osmolality. Cluster analysis grouped samples with significantly higher sperm quality and pH and significantly lower urea concentration and osmolality to indicate that urine contamination negatively affected sperm quality by increasing osmolality and decreasing pH. Amongst the six extender solutions Leibovitz and Marine Freeze® preserved significantly higher percentage motility 24 h after collection. Ringer, NAM and Stor Fish® were intermediate and Sucrose was similar to control samples that significantly decreased motility 3 h after collection. Taken together all sole sperm samples probably had urine contamination, which is difficult or impossible to avoid especially if all the sperm available needs to be collected. The extenders, Leibovitz and Marine Freeze® were used to maintain sperm quality and mitigate the negative effects of urine contamination. The collection and short term chilled storage in extenders of sole sperm from the majority of males in a broodstock (65.3%) can provide a valid sperm management system for industrial application for artificial fertilisation, however, further work is needed.info:eu-repo/semantics/acceptedVersio

Gamete quality and management for in vitro fertilisation in meagre (Argyrosomus regius)

The aquaculture of meagre (Argyrosomus regius) requires methods for the control of reproduction that enable the production of families from specific individuals for selective breeding programs. We experimentally determined the parameters required for an in vitro fertilisation protocol. A total of 14 females and 5 males (mean ± S.D. weights of 20.45 ± 6.22 and 15.94 ± 2.75 kg, respectively) were used. Selected females had vitellogenic oocytes >550 μm in diameter and males had fluid sperm upon application of abdominal pressure. Both sexes were treated with an injection of 15 μg kg−1 of gonadotropin-releasing hormone agonist (GnRHa) to induce oocyte maturation/ovulation and enhance sperm production. To determine the timing of ovulation and window of high egg viability, females were stripped serially every 2.5 h beginning 35 h after GnRHa treatment. Sperm was obtained 24 h after GnRHa treatment and was diluted 1/4 in modified Leibovitz for storage at 4 °C until use. Sperm quality parameters such as percentage initial spermatozoa motility, duration of motility, velocity and density were determined using computer assisted sperm analysis (CASA). In vitro inseminations were made in duplicate or triplicate batches of eggs from each spawn by mixing 0.5–1 mL of eggs, 20–40 μL diluted sperm (pooled from two males) and 100 mL of seawater. Fertilisation success was examined at spermatozoa (spz): egg ratios between ~2000 and 400,000 spz egg−1. The optimal time for stripping ovulated females was ≤3 h after ovulation, which was the window of optimal egg viability. Ovulation under the conditions of this study was close to 38 h after GnRHa treatment, with a range from 35 to 41 h. Beginning from 3 h after ovulation, egg viability declined probably due to overripening. Sperm diluted in Leibovitz maintained motility and velocity for as long as 7 h after collection. Spermatozoa motility (%) and average path velocity (VAP, μm/s) of sperm samples obtained from males before GnRHa injection declined rapidly after activation compared to the samples obtained 24 h post-injection, with significant decreases respectively after 75 and 45 s. A minimum ratio of 150,000 spermatozoa egg−1 was necessary to ensure high fertilisation success. The acquired knowledge of the present study will aid the aquaculture industry and future research on selective breeding programs for meagre.info:eu-repo/semantics/acceptedVersio

Transcriptome analysis of flathead grey mullet (Mugil cephalus) ovarian development induced by recombinant gonadotropin hormones

Background: Treatment with recombinant gonadotropin hormones (rGths), follicle-stimulating hormone (rFsh) and luteinizing hormone (rLh), was shown to induce and complete vitellogenesis to finally obtain viable eggs and larvae in the flathead grey mullet (Mugil cephalus), a teleost arrested at early stages of gametogenesis in intensive captivity conditions. This study aimed to investigate the transcriptomic changes that occur in the ovary of females during the rGths-induced vitellogenesis. Methods: Ovarian samples were collected through biopsies from the same five females at four stages of ovarian development. RNASeq libraries were constructed for all stages studied, sequenced on an Illumina HiSeq4000, and a de novo transcriptome was constructed. Differentially expressed genes (DEGs) were identified between stages and the functional properties of DEGs were characterized by comparison with the gene ontology and Kyoto Encyclopedia. An enrichment analysis of molecular pathways was performed. Results: The de novo transcriptome comprised 287,089 transcripts after filtering. As vitellogenesis progressed, more genes were significantly upregulated than downregulated. The rFsh application induced ovarian development from previtellogenesis to early-to-mid-vitellogenesis with associated pathways enriched from upregulated DEGs related to ovarian steroidogenesis and reproductive development, cholesterol metabolism, ovarian growth and differentiation, lipid accumulation, and cell-to-cell adhesion pathways. The application of rFsh and rLh at early-to-mid-vitellogenesis induced the growth of oocytes to late-vitellogenesis and, with it, the enrichment of pathways from upregulated DEGs related to the production of energy, such as the lysosomes activity. The application of rLh at late-vitellogenesis induced the completion of vitellogenesis with the enrichment of pathways linked with the switch from vitellogenesis to oocyte maturation. Conclusion: The DEGs and enriched molecular pathways described during the induced vitellogenesis of flathead grey mullet with rGths were typical of natural oogenesis reported for other fish species. Present results add new knowledge to the rGths action to further raise the possibility of using rGths in species that present similar reproductive disorders in aquaculture, the aquarium industry as well as the conservation of endangered species.info:eu-repo/semantics/publishedVersio

Low sperm to egg ratio required for successful in vitro fertilization in a pair-spawning teleost, Senegalese sole (Solea senegalensis)

Cultured Senegalese sole (Solea senegalensis) breeders fail to spawn fertilized eggs. The implantation of large-scale in vitro fertilization protocols, to solve this problem, has been frustrated by low production of poor quality sperm. Cultured females were induced to ovulate with a 5 µg kg−1 single injection of gonadotropin-releasing hormone agonist (GnRHa) and viable eggs (82.6 ± 9.2% fertilization) were stripped 41:57 ± 1:46 h after the injection. Sperm was collected from cultured males, diluted in modified Leibovitz and used fresh to fertilize the eggs. Males were not treated with hormones. A nonlinear regression, an exponential rise to a maximum (R = 0.93, p < 0.0001) described the number of motile spermatozoa required to fertilize a viable egg and 1617 motile spermatozoa were sufficient to fertilize 99 ± 12% (±95% CI) of viable eggs. Similar, spermatozoa egg−1 ratios of 592 ± 611 motile spermatozoa egg−1 were used in large-scale in vitro fertilizations (190 512 ± 38 471 eggs). The sperm from a single male (145 ± 50 µl or 8.0 ± 6.8 × 108 spermatozoa) was used to fertilize the eggs. The mean hatching rate was 70 ± 14% to provide 131 540 ± 34 448 larvae per fertilization. The viability of unfertilized eggs stored at room temperature decreased gradually, and the sooner eggs were fertilized after stripping, the higher the viability of the eggs. The collection of sperm directly into a syringe containing modified Leibovitz significantly increased the percentage of motile spermatozoa (33.4 ± 12.2%) compared with other collection methods. The spz egg−1 ratios for Senegalese sole were at the lower end of ratios required for fish. Senegalese sole have a pair-spawning reproductive behaviour characterized by gamete fertilization in close proximity with no sperm competition. The provision of a large-scale in vitro fertilization protocol (200 µl of sperm per 100 ml of eggs) will enable the industry to operate sustainably and implement breeding programmes to improve production.publishedVersio

Low sperm to egg ratio required for successful in vitro fertilization in a pair-spawning teleost, Senegalese sole (Solea senegalensis)

Cultured Senegalese sole (Solea senegalensis) breeders fail to spawn fertilized eggs. The implantation of large-scale in vitro fertilization protocols, to solve this problem, has been frustrated by low production of poor quality sperm. Cultured females were induced to ovulate with a 5 µg kg−1 single injection of gonadotropin-releasing hormone agonist (GnRHa) and viable eggs (82.6 ± 9.2% fertilization) were stripped 41:57 ± 1:46 h after the injection. Sperm was collected from cultured males, diluted in modified Leibovitz and used fresh to fertilize the eggs. Males were not treated with hormones. A nonlinear regression, an exponential rise to a maximum (R = 0.93, p < 0.0001) described the number of motile spermatozoa required to fertilize a viable egg and 1617 motile spermatozoa were sufficient to fertilize 99 ± 12% (±95% CI) of viable eggs. Similar, spermatozoa egg−1 ratios of 592 ± 611 motile spermatozoa egg−1 were used in large-scale in vitro fertilizations (190 512 ± 38 471 eggs). The sperm from a single male (145 ± 50 µl or 8.0 ± 6.8 × 108 spermatozoa) was used to fertilize the eggs. The mean hatching rate was 70 ± 14% to provide 131 540 ± 34 448 larvae per fertilization. The viability of unfertilized eggs stored at room temperature decreased gradually, and the sooner eggs were fertilized after stripping, the higher the viability of the eggs. The collection of sperm directly into a syringe containing modified Leibovitz significantly increased the percentage of motile spermatozoa (33.4 ± 12.2%) compared with other collection methods. The spz egg−1 ratios for Senegalese sole were at the lower end of ratios required for fish. Senegalese sole have a pair-spawning reproductive behaviour characterized by gamete fertilization in close proximity with no sperm competition. The provision of a large-scale in vitro fertilization protocol (200 µl of sperm per 100 ml of eggs) will enable the industry to operate sustainably and implement breeding programmes to improve production.info:eu-repo/semantics/publishedVersio

Gonadotropin induction of spermiation in Senegalese sole: Effect of temperature and stripping time

11 pages, 7 figures, 1 tableTreatments with homologous recombinant follicle-stimulating and luteinizing hormones (rFsh and rLh, respectively) are known to enhance spermatogenesis and sperm production in sole, but the response can be highly variable depending on the dose, duration and time of the year of the rFsh treatment. To further investigate the physiological effects of rFsh and rLh on sperm production in sole, here we examined the pattern of spermiation of F1 males, of approximately 450 g, treated with rFsh and rLh under controlled temperature. In an initial trial at 12 °C, males were weekly injected intramuscularly with 18 μg kg−1 rFsh over five weeks and subsequently treated with a single injection of 18 μg kg−1 rLh. Histological analysis indicated that the rFsh+rLh treatment increased gonad weight and stimulated spermatogenesis, and also enlarged the size of the seminiferous and efferent duct (ED) tubules, resulting in a doubling of sperm production with respect to the controls. Sperm counts in the ED and sequential stripping of males at 24, 48 and 72 h post rLh injection further revealed that only one batch of spermatids is recruited into spermatozoa (Spz) differentiation after a single rLh induction. A peak of sperm accumulation in the ED occurs at 48 h, coinciding with the upregulation of genes potentially involved in Spz maturation. In a second experiment, we tested the effect of two rFsh doses (10 or 18 μg kg−1) over five weeks as previously, followed by one rLh injection at 12 °C or 17 °C. The results confirmed that spermiation was the highest 48 h after rLh treatment at 12 °C, which was increased in a dose-dependent manner with the dose of rFsh previously supplied (from 0.36 to 0.95 × 109 Spz kg−1). However, sperm production elicited with the low rFsh dose was potentiated by ~3-fold (from 0.36 to 1.06 × 109 Spz kg−1) when the rLh treatment was given at 17 °C. These data suggest that in Senegalese sole sperm collection should be carried out at 48 h after rLh treatment, and that a low dose of rFsh at 12 °C is highly efficient for stimulating sperm production when rLh is administered at a temperature close to that occurring during maximum natural spermiationThis work was supported by the Spanish Ministry of Science and Innovation (MINECO) (AGL2017-84013-R to F.C.), the European Commission Portugal 2020 Program (European Regional Development Fund, 038433_REARLING to I.B.), and the CERCA Programme (Government of Catalonia). Participation of F.C. was funded by a “Ramon y Cajal” contract (RYC-2015-17103, MINECO). Participation of S.R. and W.G. was funded, respectively, by a PhD grant from AGAUR Catalonian Agency for the Management of Universities and Research Grants (Government of Catalonia, Spain) co-financed by the European Social Fund, and a PhD grant from the National Board of Science and Technology (CONACYT, Mexico)Peer reviewe

Control of reproduction in the flathead grey mullet Mugil cephalus : Broodstock management, hormonal therapies and transcriptomic signature

La llisa (Mugil cephalus) és una bona candidata per a la diversificació de les espècies europees cultivades per la seva adaptabilitat a diferents condicions de cultiu, alimentació omnívora i perfil ecològic. El desenvolupament futur depèn, en part, del control reproductiu en condicions intensives, necessari per proporcionar alevins criats en vivers per tal que les industries no depenguin de la captura d'alevins o de la inducció d'individus salvatges madurs, pràctiques insostenibles a llarg termini. Per tant, la present tesi s'ha centrat en aspectes de la gestió dels reproductors i en tractaments hormonals per superar la disfunció reproductiva d'aquesta espècie en captivitat. Durant els tractaments hormonals (agost - novembre), peixos salvatges i originaris d'una granja semi-extensiva mantinguts en condicions intensives -de tres mesos fins a 3,5 anys-, presentaven oòcits previtel·logènics o un percentatge baix (<10%) en vitel·logènesi primerenca, no tenien esperma o en produïen una gota viscosa. Contràriament, les femelles salvatges (n = 44) del Mediterrani occidental eren a vitel·logènesi, essent la posta al setembre i l'octubre, d'acord amb l'examen macroscòpic, histològic i biomètric realitzat. Per identificar els requisits nutricionals dels reproductors, es van examinar els canvis en la composició de lípids i d'àcids grassos del múscul, fetge i ovari de femelles salvatges en (i) previtel·logènesi (n = 7), (ii) vitel·logènesi primerenca (n = 6), (iii) vitel·logènesi tardana (n = 7) i (iv) després de la posta (n = 7). El contingut total de lípids va ser constant i baix al múscul (3,85 - 4,92 %), alt al fetge (18,46 - 22,62 %), i va augmentar durant el desenvolupament gonadal (4,90 - 34,59 %) disminuint després de la posta. Durant la vitel·logènesi, es va produir la mobilització d'àcids grassos poliinsaturats (AGPI) n-3 del fetge. El percentatge d'àcids grassos saturats, principalment 16:0, i d'AGPI, principalment dels àcids eicosapentaenoic, docosahexaenoic i araquidònic, va disminuir significativament als ovaris. En canvi, els àcids grassos monoinsaturats, principalment 16:1 i 17:1 d'origen bacterià, van augmentar significativament. L'estudi de les respostes dels peixos als pellets (2, 4, 6, 8 mm de diàmetre) i l'avaluació de l'àrea d'alimentació van indicar que els diàmetres òptims pels juvenils (~ 360 g) eren de 2 i 4 mm, mentre pels adults (~ 930 g) era de 4 mm, i que preferien alimentar-se a la columna d'aigua i al fons, enlloc de la superfície. El tractament a llarg termini amb injeccions setmanals d'hormona fol·liculostimulant recombinant (rFsh) (dosis de 4 a 15 µg kg-1) i/o d'hormona luteïnitzant (rLh) (dosis de 2,5 a 24 µg kg-1) produïdes a les cèl·lules CHO (Ovari de Hàmster Xinès) va induir i completar la vitel·logènesi al 100 % de femelles (n = 29) amb el tractament complet. El 100% dels mascles (n = 13) van produir esperma. La dosi inicial de 30 µg kg-1 rLh va induir la maduració dels oòcits 24 h rere l'administració. Les dosis resolutives de 40 mg kg-1 progesterona (n = 9 femelles) i 30 µg kg-1 rLh (n = 6) van donar lloc, respectivament, a un 89 % i 100 % d'èxit de posta amb un 63 ± 21 % de fertilització i un 58 ± 23 % d'eclosió. L'expressió diferencial de gens i vies moleculars entre les diferents etapes de la vitel·logènesi induïdes amb rFsh i rLh, avaluades després de realitzar la seqüenciació del transcriptoma (RNAseq) i l'assemblatge "de novo", van mostrar ser típiques de l'oogènesi natural en peixos. Les troballes d'aquesta tesi han permès controlar la reproducció de la llisa en condicions de cultiu intensiu, i plantegen la possibilitat d'utilitzar el tractament amb rFsh i rLh per induir la posta fora de temporada i en altres espècies amb trastorns reproductius similars.El múgil (Mugil cephalus) es un buen candidato para la diversificación de las especies europeas cultivadas por su adaptabilidad a diferentes condiciones de cultivo, alimentación omnívora y perfil ecológico. El desarrollo futuro depende, en parte, del control reproductivo en condiciones intensivas, necesario para proporcionar alevines criados en viveros para que las industrias no dependan de la captura de alevines o de la inducción de individuos salvajes maduros, prácticas insostenibles a largo plazo. Por lo tanto, la presente tesis se ha centrado en aspectos de la gestión de los reproductores y en tratamientos hormonales para superar la disfunción reproductiva de esta especie en cautividad. Durante los tratamientos hormonales (agosto - noviembre), peces salvajes y originarios de una granja semi-extensiva mantenidos en condiciones intensivas -de tres meses a 3,5 años-, presentaban ovocitos previtelogénicos o un porcentaje bajo (<10%) en vitelogénesis temprana, no tenían esperma o producían una gota viscosa. Contrariamente, las hembras salvajes (n = 44) del Mediterráneo occidental estaban en vitelogénesis, siendo el desove en septiembre y octubre, de acuerdo con el examen macroscópico, histológico y biométrico realizado. Para identificar los requisitos nutricionales de los reproductores, se examinaron los cambios en la composición de lípidos y ácidos grasos del músculo, hígado y ovario de hembras salvajes en (i) previtelogénesis (n = 7), (ii) vitelogénesis temprana (n = 6), (iii) vitelogénesis tardía (n = 7) y (iv) tras la puesta (n = 7). El contenido total de lípidos fue constante y bajo en el músculo (3,85 - 4,92%), alto en el hígado (18,46 - 22,62%), y aumentó durante el desarrollo gonadal (4,90 - 34,59%) disminuyendo tras la puesta. Durante la vitelogénesis, se produjo la movilización de ácidos grasos poliinsaturados (AGPI) n-3 del hígado. El porcentaje de ácidos grasos saturados, principalmente 16:0, y de AGPI, principalmente de los ácidos eicosapentaenoico, docosahexaenoico y araquidónico, disminuyó significativamente en los ovarios. En cambio, los ácidos grasos monoinsaturados, principalmente 16:1 y 17:1 de origen bacteriano, aumentaron significativamente. El estudio de las respuestas de los peces a los pellets (2, 4, 6, 8 mm de diámetro) y la evaluación del área de alimentación indicaron que los diámetros óptimos para juveniles (~ 360 g) eran de 2 y 4 mm, mientras para adultos (~ 930 g) era de 4 mm, y que preferían alimentarse en la columna de agua y en el fondo, en vez de en la superficie. El tratamiento a largo plazo con inyecciones semanales de hormona foliculoestimulante recombinante (rFsh) (dosis de 4 a 15 µg kg-1) y/o de hormona luteinizante (rLh) (de 2,5 a 24 µg kg-1) producidas en células CHO (Ovario de Hámster Chino) indujo y completó la vitelogénesis en el 100 % de hembras (n = 29) con el tratamiento completo. El 100% de machos (n = 13) produjeron esperma. La dosis inicial de 30 µg kg-1 rLh indujo la maduración de los ovocitos 24 h después. Las dosis resolutivas de 40 mg kg-1 progesterona (n = 9 hembras) y 30 µg kg-1 rLh (n = 6) dieron lugar, respectivamente, a un 89 % y 100 % de desoves con 63 ± 21% de fertilización y 58 ± 23% de eclosión. La expresión diferencial de genes y vías moleculares entre los diferentes estados vitelogénicos inducidos con rFsh y rLh, evaluada tras realizar la secuenciación del transcriptoma (RNAseq) y el ensamblaje "de novo", mostraron ser típicas de la ovogénesis natural en peces. Los hallazgos de esta tesis han permitido controlar la reproducción del múgil en cultivo intensivo, y plantean la posibilidad de utilizar el tratamiento con rFsh y rLh para inducir el desove fuera de temporada y en otras especies con trastornos reproductivos similares.The flathead grey mullet (Mugil cephalus) is a good candidate for the diversification of European species in aquaculture due to characteristics such as, adaptability to different culture conditions, omnivorous feeding and ecological profile. The future development partly relies on the control of reproduction in intensive conditions, which is needed to provide hatchery-reared fry to end the industries dependence on wild fry or the induction of wild mature individuals, which are unsustainable in the long term. Therefore, the present thesis has focused on several aspects related to broodstock management and on hormonal treatments to overcome the reproductive dysfunction this species presents in captivity. The present thesis found that, during hormonal treatments (August - November), wild fish and fish from a semi-extensive farm held in captive conditions - from three months to 3.5 years-, presented only previtellogenic oocytes or had a low percentage (<10 %) of early-vitellogenic oocytes, had no milt or produced a viscous drop. Contrarily, wild females (n = 44) in the Western Mediterranean had started vitellogenesis, and between September and October spawned, according to macroscopic, histological, and biometric examination. To identify the broodstock nutritional requirements, changes in lipid and fatty acid composition of muscle, liver and ovary of wild flathead grey mullet females at (i) previtellogenesis (n = 7), (ii) early-vitellogenesis (n = 6), (iii) late-vitellogenesis (n =7), and (iv) post-spawning period (n = 7) were characterized. Total lipid content was constant and low in the muscle (3.85 - 4.92 %), high in the liver (18.46 - 22.62 %), and increased during gonadal development (4.90 - 34.59 %) to decrease after spawning. During vitellogenesis, there was a mobilization of n-3 polyunsaturated fatty acids (PUFA) from the liver. Percentage of total saturated fatty acids deposits, mainly of palmitic acid (16:0), and of PUFA, mainly of eicosapentaenoic (EPA), docosahexaenoic (DHA), and arachidonic (ARA) acids, significantly decreased in the ovaries. Contrarily, mono-unsaturated fatty acids, principally of palmitoleic acid (16:1) and heptadecenoic acid (17:1), which is of bacterial origin, significantly increased. The study of the behavioural responses to pellets (2, 4, 6, 8 mm diameter) and the preferred feeding area indicated that optimal pellet diameters for juveniles (~ 360 g) were 2 and 4 mm, while for adults (~ 930 g) was 4 mm, and that flathead grey mullet preferred to feed in the water column and the bottom rather than in the surface. Long-term treatment with weekly injections of single-chain recombinant follicle- stimulating hormone (rFsh) (4 to 15 µg kg-1 doses) and /or recombinant luteinizing- hormone (rLh) (2.5 to 24 µg kg-1 doses) produced in CHO (Chinese Hamster Ovary) cells induced and completed vitellogenesis in 100 % of females (n = 29) that received the complete treatment. The 100 % of males (n = 13) spermiated. Priming dose of 30 µg kg- 1 rLh induced oocyte maturation 24 h after administration. Resolving dose of 40 mg kg-1 Progesterone (n = 9 females) and 30 µg kg-1 rLh (n = 6) resulted in 89 % and 100 % spawning success, respectively, with 63 ± 21 % fertilization and 58 ± 23 % hatching. The differential expression of genes and enriched molecular pathways amongst different stages of vitellogenesis induced with the rFsh and rLh treatment, after RNA-Seq and de novo transcriptome assembly, were typical of natural oogenesis reported for other fish species. The findings from this thesis have permitted to control the reproduction of flathead grey mullet in intensive culture conditions, and raise the possibility of using the rFsh and rLh treatment to develop out-of-season spawning and apply it in other species that present similar reproductive disorders

Lipid and fatty acid composition of muscle, liver, ovary, and peritoneal fat in wild flathead grey mullet (Mugil cephalus) according to ovarian development

Wild adult females of a low trophic omnivore teleost species, the flathead grey mullet (Mugil cephalus), caught in the western Mediterranean were sampled. The lipid and fatty acid composition of ovaries, liver, muscle, and peritoneal fat were analysed at previtellogenesis, early-vitellogenesis —first observed at mid-summer (early August)—, late-vitellogenesis, and the post-spawning period —from mid-September to mid-October—. During ovarian development, the lipid content of muscle was low and constant (3.85%–4.92%), indicating that the muscle was not used to store lipids for gonadal growth. Although constant, lipid content in the liver was higher (18.46%–22.62%) than in the muscle, and HSI% increased during gonad development, suggesting a dynamism in the mobilization of the hepatic lipids. Total lipids in the gonads significantly increased with maturation (from 4.90% to 34.59%) in parallel with the GSI (from 0.8% to 15.5%) to decrease after spawning. Peritoneal fat was probably transitional fat that could be rapidly metabolized or transferred to other tissues but no specific function could be assigned because its presence in previtellogenic and early-vitellogenic females varied greatly. One of the main sources of lipids accumulated in the ovary was most likely diet. The total percentage of ΣMUFA, mainly 17:1 —previously not identified in high quantities in teleost vitellogenic ovaries and likely of bacterial origin— and 16:1, strongly increased in the ovaries with maturation. The 16:1 might be an important source of lipids for embryo development. High percentages of DHA, EPA, and ARA were found in the ovary during previtellogenesis available to be used during gonadal maturation. Understanding lipid and fatty acid changes in broodstock tissues can increase our knowledge of the nutritional requirements of the fish used in aquaculture breeding programs.info:eu-repo/semantics/acceptedVersio

Reproductive behaviour and fertilized spawns in cultured Solea senegalensis broodstock co-housed with wild breeders during their juvenile stages

The reproductive failure of Senegalese sole (Solea senegalensis) cultured males (reared entirely in captivity from egg through to adult) that do not participate in reproductive behaviours to fertilise spawns, results in a problem to achieve reproductive control in captivity. However, cohabitation with wild males has led to an increase in the involvement of cultured males in reproductive behaviour, although their contribution to fertilised spawning is still lower than that of wild breeders. This study aimed to examine the effect of different social conditions, on the reproductive behaviour and spawning success of cultured breeders over three reproductive seasons. Before starting this study, different social learning opportunities were provided to the breeders from the juvenile to the pubertal stages of the individuals. Behaviour and spawning were evaluated in four experimental groups of cultured breeders: two groups (W1 and W2) that prior to this study were reared during the juvenile stage with wild breeders that fertilized spawns, a Culture breeder group (CB) that was previously reared with cultured breeders that spawned unfertile eggs, and a negative control group (CN) that was reared in isolation from adult fish. During the three reproductive seasons, spawning was obtained from all groups. Generally, the first year had the highest egg production and the third year the lowest. However, fertilised eggs were only obtained from W1 in the first year. A total of eight fertilised spawns were collected with a fertilisation rate of 28.02 ± 13.80 % and a hatching rate of 15.04 ± 10.40 %. The mean number of larvae obtained per spawn was 7,683 ± 5,947 and the total number of larvae from all eight spawns was 61,468. The paternity analysis assigned 64.3 % of larvae to a single couple of breeders, while 34.3 % of larvae were not assigned to any single family, but inconclusively to more than three parents. The highest locomotor activity was observed in W1, while no significant differences were observed in the number of movements within W2, CB and CN. In all groups, during the peak of locomotor activity (19h00-20h00), the main reproductive behaviours observed were Rest the Head and Follow, while the Guardian behaviour was low and Coupled behaviour was only observed in W1. Over time, the reproductive behaviours decreased, except for Follow. The social learning opportunities provided by cohabitation with wild fish during juvenile stages prior to spawning in W1, increased activity and fertilised spawning. However, the number of successful spawns was low and over time stopped in association with a decrease in reproductive behaviour. This suggests that other mechanisms of behavioural learning could be involved in reproductive success, such as reproductive dominance, environmental conditions or hormonal interactions that could affect physiological processes in the reproduction of captive breeders.The authors would like to thank Josep Lluis Celades and Marta Sastre for technical help. This work was funded by the National Institute of Agricultural Research and Technology and Food INIA-FEDER (RTA2014-00048) coordinated by ND. Participation of SR was supported by a PhD grant from AGAUR (Government of Catalonia) and WG was funded by a predoctoral grant from the National Board of Science and Technology (CONACYT, México).info:eu-repo/semantics/acceptedVersio