Effect of prolonged feeding of broodstock diet with increased inclusion of essential n-3 fatty acids on maturing and spawning performance in 3-year-old Atlantic salmon (Salmo salar).

Atlantic salmon (Salmo salar) broodstock recruits are normally fed a specialized diet with a higher content of essential nutrients for a limited time period prior to fasting and transfer to freshwater. Typically, this period lasts for about six months, but may vary among producers. Reduced use of marine ingredients in commercial salmon diets during the last decades has affected the content of essential nutrients, such as n-3 long chained polyunsaturated fatty acids (LC-PUFA), minerals and vitamins. Furthermore, to minimize the risk of losses and implement new breeding achievements faster, breeding companies have shortened the production cycle of broodstock from 4 to 3 years, which may affect the number of fish that are large enough to mature. In the present study, we have extended the broodstock feeding period from 6 to 15 months prior to the freshwater transfer giving a higher content of n-3 LC-PUFA (higher inclusion of marine oils) from February to December (Phase 1), and thereafter a diet with a higher energy content to ensure growth towards the spring and maturation (Phase 2). Four sea cages with approximately 80.000 salmon postsmolt, two sea cages with males and two with females, were given a control diet and an experimental diet. Samples were taken in Phase 1 at start (1.7 kg), mid (3.4 kg) and end Phase 1/start of Phase 2 (8.3 kg), and end of Phase 2 (13.4 kg). The fish were thereafter fasted, and selected fish transferred to landbased freshwater tanks where light and temperature were used to manipulate the spawning time of the fish in two groups (early or late). Due to disease in the facility, measures of egg quality and hatching were only obtained from the early group. During the trial and spawning period, biometrical measurements were recorded, and samples of liver, gonad, fillet and red blood cells (RBC) were collected for fatty acid composition and blood plasma for analysis of lipid and health-related parameters. Samples were also collected for gonadal transcriptomic analysis by microarray and qPCR (end Phase 2) and plasma steroids (end Phase 2, mid maturation and spawning). Males fed the test diet had a larger body size compared to the control group at the end of Phase 2, while no differences were observed between dietary groups for the females. Total mortality in the trial was lower in the test group compared to the control, losses were caused mainly by sea lice treatments, loser fish or cardiomyopathy syndrome (CMS). The dietary LC-PUFA levels in the test diet were reflected in the tissues particularly during Phase 1, but only different in the fillet samples and eggs at the end of Phase 2 and at spawning. Plasma sex steroids content increased at mid maturation and showed lower levels of androgens and estrogens in females fed the test diet compared to the control. At the end of Phase 2, transcriptional analysis showed upregulation of steroidogenic enzymes, although not reflected in changes in plasma steroids in Phase 2, indicating changes to come during maturation. The differences in LC-PUFA content in tissues and plasma steroids did not appear to affect fecundity, sperm quality, egg survival or hatching rate, but the test group had larger eggs compared to the control in the early spawner-group. Prolonged feeding of n-3 LC-PUFA to pre-puberty Atlantic salmon broodstock appears to be important for higher survival in challenging sea cage environments and has an effect on sex steroid production that, together with high energy diet during early maturation, cause the test group to produce larger eggs.

Benzodiazepine Interference with Fertility and Embryo Development: A Preliminary Survey in the Sea Urchin Paracentrotus lividus.

Psychotropic drugs and benzodiazepines are nowadays among the primary substances of abuse. This results in a large and constant release into aquatic environments where they have potentially harmful effects on non-target organisms and, eventually, human health. In the last decades, evidence has been collected on the possible interference of benzodiazepines with reproductive processes, but data are few and incomplete. In this study, the possible negative influence of delorazepam on fertilization and embryo development has been tested in Paracentrotus lividus, a key model organism in studies of reproduction and embryonic development. Sperm, eggs, or fertilized eggs have been exposed to delorazepam at three concentrations: 1 µg/L (environmentally realistic), 5 µg/L, and 10 µg/L. Results indicate that delorazepam reduces the fertilizing capacity of male and female gametes and interferes with fertilization and embryo development. Exposure causes anatomical anomalies in plutei, accelerates/delays development, and alters the presence and distribution of glycoconjugates such as N-Acetyl-glucosamine, α-linked fucose, and α-linked mannose in both morulae and plutei. These results should attract attention to the reproductive fitness of aquatic species exposed to benzodiazepines and pave the way for further investigation of the effects they may exert on human fertility. The presence of benzodiazepines in the aquatic environment raises concerns about the reproductive well-being of aquatic species. Additionally, it prompts worries regarding potential impacts on human fertility due to the excessive use of anxiolytics.

Effects of Di-(2-Ethylhexyl) Phthalate (DEHP) on Gamete Quality Parameters of Male Koi Carp (Cyprinus carpio).

In this study, we evaluated gamete quality parameters of mature male koi carp (Cyprinus carpio) exposed to three different concentrations (1, 10, and 100 µg/L) of di-(2-ethylhexyl) phthalate (DEHP). After 60 days of exposure, there was a significant decrease in the gonadosomatic index (GSI) of males exposed to 10 and 100 µg/L of DEHP. Histological analysis of the testes revealed impaired histoarchitecture, including inflammatory cells, intratubular vacuoles, and swollen seminiferous tubules in treatment groups. Gamete quality parameters like sperm production, motility, spermatocrit, and sperm density values were significantly decreased at the 10 and 100 µg/L concentrations. Biochemical compositions, including glucose, cholesterol, and total protein levels, were significantly changed in the treatment groups. Similarly, the ionic compositions of seminal fluid (Na, K, Ca, and Mg) also varied in the treatment groups. Furthermore, the 11-ketotestosterone levels were decreased, and the 17-ß estradiol levels were increased in the DEHP-treated groups. The mRNA expression levels of reproduction-related genes, including Fshr, Lhr, Ar, Erα, and Erß, were significantly changed in the DEHP-treated males in a dose-dependent manner. In conclusion, the findings of this study confirmed that environmentally relevant exposure to DEHP may contribute to a decline in the gamete quality of male fishes.

Broodstock exposure to warming and elevated pCO2 impairs gamete quality and narrows the temperature window of fertilisation in Atlantic cod.

Impacts of global warming and CO2 -related ocean acidification (OA) on fish reproduction may include chronic effects on gametogenesis and gamete quality, as well as acute effects on external fertilisation. Here, temperature thresholds and OA-sensitivity of gametogenesis and fertilisation were investigated in Atlantic cod, Gadus morhua. Three broodstock groups of farmed cod (FC 1-3) were exposed for 3 months to three maturation conditions (FC 1: control, 6°C/400 µatm CO2 ; FC 2: warming, 9.5°C/400 µatm; FC 3: warming and OA, 9.5°C/1100 µatm). In addition, a broodstock group of wild cod (WC) was kept at control conditions to compare the acute temperature window of fertilisation with that of farmed cod (FC 1). Fertilisations were conducted in a temperature-gradient table at 10 temperatures (between -1.5 and 12°C) and two CO2 levels (400/1100 µatm). In FC 1 and WC, fertilisation success was relatively high between 0.5°C and 11°C (TRange of c. 10.5°C), indicating similar gamete quality in farmed and wild broodstocks kept at control conditions. Exposure of farmed broodstocks to warming (FC 2) and the combination of warming and OA (FC 3) impaired gamete quality, causing a reduction in fertilisation success of -20% (FC 2) and - 42% (FC 3) compared to FC 1. The acute temperature window of fertilisation narrowed from FC 1 (TRange  = 10.4°C) to FC 2 (TRange  = 8.8°C) and FC 3 (TRange  = 5.9°C). Acute effects of CO2 on fertilisation success were not significant. This study demonstrates potential climate change impacts on gametogenesis and fertilisation in Atlantic cod, suggesting the loss of spawning habitat in the coming decades.

Insights on the seasonal variations of reproductive features in the Eastern Atlantic Bluefin Tuna.

The Atlantic Bluefin Tuna (ABFT, Thunnus thynnus) is one of the most intensely exploited fisheries resources in the world. In spite of the years of studies on ABFT, basic aspects of its reproductive biology remain uncertain. To gain insight regarding the seasonal changes of the reproductive characteristics of the eastern stock of ABFT, blood and tissue samples were collected from mature specimens caught in the Mediterranean basin during the reproductive (May-June) and non-reproductive season (Oct-Nov). Histological analysis of the gonads of May-June samples indicated that there were females which were actively spawning (contained post-ovulatory follicles) and females that were not actively spawning that had previtellogenic and fully vitellogenic oocytes. In males, testis were at early or late stage of spermatogenesis during the reproductive season. In Oct-Nov, ovaries contained mostly previtellogenic oocytes as well as ß and α atretic follicles while the testis predominantly contained spermatogonia and few cysts with spermatocytes and spermatozoa. Gonadosomatic index (GSI) in females was highest among the actively spawning individuals while in males GSI was higher in early and late spermatogenic individuals compared to those that were spent. Plasma sex steroids levels varied with the reproductive season. In females, estradiol (E2), was higher in May-June while testosterone (T) and progesterone (P) did not vary. In males, E2 and T were higher in May-June while P levels were similar at the two sampling points. Circulating follicle stimulating hormone (FSH) was higher in Oct-Nov than in May-June both in males and females. Vitellogenin (VTG) was detected in plasma from both males and females during the reproductive season with levels in females significantly higher than in males. VTG was undetected in Oct-Nov samples. Since choriogenesis is an important event during follicle growth, the expression of three genes involved in vitelline envelope formation and hardening was measured and results showed significantly higher levels in ovaries in fish caught in May-June with respect to those sampled in Oct-Nov. In addition, a set of genes encoding for ion channels that are responsible for oocyte hydration and buoyancy, as well as sperm viability, were characterized at the two time points, and these were found to be more highly expressed in females during the reproductive season. Finally, the expression level of three mRNAs encoding for different lipid-binding proteins was analyzed with significantly higher levels detected in males, suggesting sex-specific expression. Our findings provide additional information on the reproductive biology of ABFT, particularly on biomarkers for the assessment of the state of maturation of the gonad, highlighting gender-specific signals and seasonal differences.

Flow cytometric assessment of morphology, viability, and production of reactive oxygen species of Crassostrea gigas oocytes. Application to toxic dinoflagellate (Alexandrium minutum) exposure.

The Pacific oyster Crassostrea gigas accounts for a large part of shellfish aquaculture production worldwide. Aspects of morphological and functional characteristics of oyster oocytes remain poorly documented, and traditional techniques, such as microscopic observations of shape or fertilization rate, are time and space consuming. The purpose of this study was to assess for the first time viability and reactive oxygen species (ROS) production of Pacific oyster oocytes using flow cytometry (FCM) and to apply this method to determine oocyte responses to in vitro exposure to the toxic dinoflagellate Alexandrium minutum. A culture of A. minutum caused a significant increase in oocyte ROS production, which gradually increased with the age of the culture, but viability was not affected. Effect of the supernatant of the same A. minutum culture did not cause any significant modifications of oocyte morphology, viability, or ROS level. This study confirmed that some oocyte cellular characteristics can be assessed using FCM techniques.

Evolutionarily conserved ovarian fluid proteins are responsible for extending egg viability in salmonid fish.

In contrast to most fishes, salmonids exhibit the unique ability to hold their eggs for several days after ovulation without significant loss of viability. During this period, eggs are held in the body cavity in a biological fluid, the coelomic fluid (CF) that is responsible for preserving egg viability. To identify CF proteins responsible for preserving egg viability, a proteomic comparison was performed using 3 salmonid species and 3 non-salmonid species to identify salmonid-specific highly abundant proteins. In parallel, rainbow trout CF fractions were purified and used in a biological test to estimate their egg viability preservation potential. The most biologically active CF fractions were then subjected to mass spectrometry analysis. We identified 50 proteins overabundant in salmonids and present in analytical fractions with high egg viability preservation potential. The identity of these proteins illuminates the biological processes participating in egg viability preservation. Among identified proteins of interest, the ovarian-specific expression and abundance in CF at ovulation of N-acetylneuraminic acid synthase a (Nansa) suggest a previously unsuspected role. We show that salmonid CF is a complex biological fluid containing a diversity of proteins related to immunity, calcium binding, lipid metabolism, proteolysis, extracellular matrix and sialic acid metabolic pathway that are collectively responsible for preserving egg viability.

Effects of reactive oxygen species and mitochondrial dysfunction on reproductive aging.

Mitochondria, the versatile organelles crucial for cellular and organismal viability, play a pivotal role in meeting the energy requirements of cells through the respiratory chain located in the inner mitochondrial membrane, concomitant with the generation of reactive oxygen species (ROS). A wealth of evidence derived from contemporary investigations on reproductive longevity strongly indicates that the aberrant elevation of ROS level constitutes a fundamental factor in hastening the aging process of reproductive systems which are responsible for transmission of DNA to future generations. Constant changes in redox status, with a pro-oxidant shift mainly through the mitochondrial generation of ROS, are linked to the modulation of physiological and pathological pathways in gametes and reproductive tissues. Furthermore, the quantity and quality of mitochondria essential to capacitation and fertilization are increasingly associated with reproductive aging. The article aims to provide current understanding of the contributions of ROS derived from mitochondrial respiration to the process of reproductive aging. Moreover, understanding the impact of mitochondrial dysfunction on both female and male fertility is conducive to finding therapeutic strategies to slow, prevent or reverse the process of gamete aging, and thereby increase reproductive longevity.

Characterization of preovulatory follicular fluid secretome and its effects on equine oocytes during in vitro maturation.

In vitro maturation (IVM) of oocytes is clinically used in horses to produce blastocysts but current conditions used for horses are suboptimal. We analyzed the composition of equine preovulatory follicular fluid (FF) secretome and tested its effects on meiotic competence and gene expression in oocytes subjected to IVM. Preovulatory FF was obtained, concentrated using ultrafiltration with cut-off of 10 kDa, and stored at -80 °C. The metabolic and proteomic composition was analyzed, and its ultrastructural composition was assessed by cryo-transmission microscopy. Oocytes obtained post-mortem or by ovum pick up (OPU) were subjected to IVM in the absence (control) or presence of 20 or 40 µg/ml (S20 or S40) of secretome. Oocytes were then analyzed for chromatin configuration or snap frozen for gene expression analysis. Proteomic analysis detected 255 proteins in the Equus caballus database, mostly related to the complement cascade and cholesterol metabolism. Metabolomic analysis yielded 14 metabolites and cryo-transmission electron microscopy analysis revealed the presence of extracellular vesicles (EVs). No significant differences were detected in maturation rates among treatments. However, the expression of GDF9 and BMP15 significantly increased in OPU-derived oocytes compared to post-mortem oocytes (fold increase ± SEM: 9.4 ± 0.1 vs. 1 ± 0.5 for BMP15 and 9.9 ± 0.3 vs. 1 ± 0.5 for GDF9, respectively; p < 0.05). Secretome addition increased the expression of TNFAIP6 in S40 regardless of the oocyte source. Further research is necessary to fully understand whether secretome addition influences the developmental competence of equine oocytes.

Reproductive toxicity assessment of cellulose nanofibers, citric acid, and branched polyethylenimine in sea urchins: Eco-design of nanostructured cellulose sponge framework (Part B).

In the framework of a safe-by-design approach, we previously assessed the eco-safety of nanostructured cellulose sponge (CNS) leachate on sea urchin reproduction. It impaired gamete quality, gamete fertilization competence, and embryo development possibly due to the leaching of chemical additives used during the CNS synthesis process. To extend this observation and identify the component(s) that contribute to CNS ecotoxicity, in the present study, we individually screened the cytotoxic effects on sea urchin Arbacia lixula and Paracentrotus lividus gametes and embryos of the three main constituents of CNS, namely cellulose nanofibers, citric acid, and branched polyethylenimine. The study aimed to minimize any potential safety risk of these components and to obtain an eco-safe CNS. Among the three CNS constituents, branched polyethylenimine resulted in the most toxic agent. Indeed, it affected the physiology and fertilization competence of male and female gametes as well as embryo development in both sea urchin species. These results are consistent with those previously reported for CNS leachate. Moreover, the characterisation of CNS leachate confirmed the presence of detectable branched polyethylenimine in the conditioned seawater even though in a very limited amount. Altogether, these data indicate that the presence of branched polyethylenimine is a cause-effect associated with a significant risk in CNS formulations due to its leaching upon contact with seawater. Nevertheless, the suggested safety protocol consisting of consecutive leaching treatments and conditioning of CNS in seawater can successfully ameliorate the CNS ecotoxicity while maintaining the efficacy of its sorbent properties supporting potential environmental applications.

Alterations in Gene Expression and the Fatty Acid Profile Impact but Do Not Compromise the In Vitro Maturation of Zebrafish (Danio rerio) Stage III Ovarian Follicles after Cryopreservation.

The vitrification of ovarian follicles is a strategic tool that may contribute to advances in aquaculture and the conservation of many important species. Despite the difficulties inherent to the cryopreservation of oocytes, some successful protocols have been developed for different species, but little is known about the capacity of oocytes to develop after thawing. Therefore, the profiles of the reproductive pathway genes and fatty acid membrane composition during the initial stages of development were analyzed in fresh ovarian follicles and follicles after the vitrification process. There were differences in the expression of the hypothalamic-pituitary-gonad axis genes during the follicular development in the control group as well as in the vitrified group. Similarly, alterations in the composition of fatty acids were observed after vitrification. Despite this, many alterations were observed in the vitrified group; more than half of the stage III ovarian follicles were able to grow and mature in vitro. Therefore, the vitrification of ovarian follicles may impact them at molecular and membrane levels, but it does not compromise their capability for in vitro maturation, which indicates that the technique can be a strategic tool for aquaculture.

Reproductive aging: biological pathways and potential interventive strategies.

Reproductive aging is a natural process conserved across species and is well-known in females. It shows age-related follicle depletion and reduction of oocyte quality, eventually causing reproductive senescence and menopause. Although reproductive aging in males is not well noticed as in females, it also causes infertility and has deleterious consequences on the offspring. Various factors have been suggested to contribute to reproductive aging, including oxidative stress, mitochondrial defects, telomere shortening, meiotic chromosome segregation errors and genetic alterations. With the increasing trend of pregnancy age, it is particularly crucial to find interventions to preserve or extend human fertility. Studies in humans and model organisms have provided insights into the biological pathways associated with reproductive aging, and a series of potential interventive strategies have been tested. Here, we review factors affecting reproductive aging in females and males and summarize interventive strategies that may help delay or rescue the aging phenotypes of reproduction.

Nanostructured cellulose sponge engineered for marine environmental remediation: Eco-safety assessment of its leachate on sea urchin reproduction (Part A).

Nanostructured cellulose sponges (CNS) have been developed as eco-friendly and sustainable engineered materials for marine environmental remediation. Despite their functionality, sensitivity, efficiency and specificity have been proved, CNS application is still limited since their environmental safety (eco-safety) has not been completely assessed. In this study, CNS were allowed to leach in natural seawater simulating the remediation process condition and the eco-safety of CNS leachate on sea urchin reproduction has been assessed by carrying out a multi-response integrated approach, combining standardized ecotoxicity tests, innovative bioassays and gamete quality assessment. Overall, the ecotoxicity data indicate that CNS leachate affects gamete quality, gamete fertilisation competence, and embryo development probably associated with the release of chemical additives used during the synthesis process. However, in the framework of the eco-design approach, consecutive leaching treatments and conditioning of CNS in seawater open the route for a new safety protocol successfully solving the ecotoxicity while maintaining CNS sorbent properties. A safe environmental application of the resulting conditioned CNS for seawater pollution remediation is envisaged.

Gamete quality in a multistressor environment.

Over the past few decades, accumulated evidence confirms that the global environment conditions are changing rapidly. Urban industrialization, agriculture and globalization have generated water, air and soil pollution, giving rise to an environment with a growing number of stress factors, which has a serious impact on the fitness, reproduction and survival of living organisms. The issue raises considerable concern on biodiversity conservation, which is now at risk: it is estimated that a number of species will be extinct in the near future. Sexual reproduction is the process that allows the formation of a new individual and is underpinned by gamete quality defined as the ability of spermatozoa and oocytes to interact during fertilization leading to the creation and development of a normal embryo. This review aimed to provide the current state of knowledge regarding the impact of a broad spectrum of environmental stressors on diverse parameters used to estimate and evaluate gamete quality in humans and in canonical animal models used for experimental research. Effects of metals, biocides, herbicides, nanoparticles, plastics, temperature rise, ocean acidification, air pollution and lifestyle on the physiological parameters that underlie gamete fertilization competence are described supporting the concept that environmental stressors represent a serious hazard to gamete quality with reproductive disorders and living organism failure. Although clear evidence is still limited, gamete capacity to maintain and/or recover physiological conditions is recently demonstrated providing further clues about the plasticity of organisms and their tolerance to the pressures of pollution that may facilitate the reproduction and the persistence of species within the scenario of global change. Changes in the global environment must be urgently placed at the forefront of public attention, with a massive effort invested in further studies aimed towards implementing current knowledge and identifying new methodologies and markers to predict impairment of gamete quality.

When few survive to tell the tale: thymus and gonad as auditioning organs: historical overview.

Unlike other organs, the thymus and gonads generate nonuniform cell populations, many members of which perish, and a few survive. While it is recognized that thymic cells are "audited" to optimize an organism's immune repertoire, whether gametogenesis could be orchestrated similarly to favor high-quality gametes is uncertain. Ideally, such quality would be affirmed at early stages before the commitment of extensive parental resources. A case is here made that, along the lines of a previously proposed lymphocyte quality control mechanism, gamete quality can be registered indirectly through detection of incompatibilities between proteins encoded by the grandparental DNA sequences within the parent from which haploid gametes are meiotically derived. This "stress test" is achieved in the same way that thymic screening for potential immunological incompatibilities is achieved-by "promiscuous" expression, under the influence of the AIRE protein, of the products of genes that are not normally specific for that organ. Consistent with this, the Aire gene is expressed in both thymus and gonads, and AIRE deficiency impedes function in both organs. While not excluding the subsequent emergence of hybrid incompatibilities due to the intermixing of genomic sequences from parents (rather than grandparents), many observations, such as the number of proteins that are aberrantly expressed during gametogenesis, can be explained on this basis. Indeed, promiscuous expression could have first evolved in gamete-forming cells where incompatible proteins would be manifest as aberrant protein aggregates that cause apoptosis. This mechanism would later have been co-opted by thymic epithelial cells which display peptides from aggregates to remove potentially autoreactive T cells.

Zebrafish as a Model to Screen the Potential of Fatty Acids in Reproduction.

Sperm quality is an important topic in general health, chemotherapy, and gamete preservation technology. Fatty acid (FA) composition of membranes, which is influenced by the diet, plays key roles in sperm biology and quality. Dietary supplementation with natural products can be used as a technique to screen potential agents to protect, modify, and recover sperm quality. In this study, zebrafish (male [♂-ZF] and female [♀-ZF]) were fed a single cultivar olive oil (OO) bioencapsulated in Artemia. OO-treated ♂-ZF had higher (p < 0.05) sperm density and motility compared to the Artemia nauplii (AN). A significant difference was also observed in follicle abundance at different stages of gametogenesis, and a nonsignificant increase in total fecundity between OO-treated ♀-ZF and the AN, although in OO-treated ♀-ZF, mature follicles had a smaller diameter. A higher fertility rate (FR) was observed in OO-treated pairs compared to the other groups. Hatching in the OO-treated fish was accelerated, although no significant differences could be found in terms of hatching rate (HR) and embryo/larval survival rate (SR). These findings in FR, HR, and SR were also confirmed in male and female replacement mating trials. Taken together, this study shows that altering the FA ratios in the diet has a clear impact on several reproductive parameters in the zebrafish, adding new information about the nutritional requirement of this model species.

Hormonal induction of ovulation using Ovaprim™ [(D-Arg6, Pro9NEt)-sGnRH+domperidone] and its impact on embryonic development of wild-caught Longspine scraper, Capoeta trutta (Heckel, 1843).

Knowledge of gamete quality is a prerequisite for developing techniques to fertilize eggs and rear offspring for hatchery production. Our objective was to develop assisted reproductive techniques, via hormonal induction of final oocyte maturation (FOM), for Longspine scraper, Capoeta trutta. Fish were administered injections of salmon gonadotropin-releasing hormone analogue containing anti-dopaminergic drug (Ovaprim™) or saline (control). Effects of Ovaprim on induction of ovulation, gamete quality, embryonic development, and larval survival were later examined with serum steroid hormone levels and ovarian histology. The saline group failed to spawn, whereas Ovaprim accelerated FOM and induced spawning. Fish treated with Ovaprim showed an increase in gonadosomatic index, egg diameter, and wet weight relative to controls. Average absolute fecundity, relative fecundity, fertilization, and hatching rates were 8823 eggs/spawn, 53 eggs/g body weight, 95%, and 91%, respectively. Serum 17α,20ß-dihydroxy-4-pregnen-3-one (DHP) levels were significantly enhanced by ∼4-fold in Ovaprim-treated fish compared to the saline-injected fish, while 17ß-estradiol levels declined upon FOM in hormone treated fish. Embryonic development closely resembled the teleost scheme, despite variations in timing. Larval survival at 6 and 12days post-hatch were 98% and 95%, respectively. Results suggest that Ovaprim is efficient for inducing spawning in C. trutta for stock enhancement or hatchery purposes.

Pikeperch Sander lucioperca egg quality cannot be predicted by total antioxidant capacity and mtDNA fragmentation.

In farmed pikeperch, there is a high variability in egg quality restraining the propagation of this species in aquaculture. The identification of reliable biomarkers for predicting successful embryo development already at an early stage (unfertilized oocyte) could help improve production efficiency. Total antioxidant capacity (TAC) and the quantification of mitochondrial DNA (mtDNA) fragmentation have been established as biomarkers for oxidative stress and damage of macromolecules, potentially influencing embryo development. Therefore, we evaluated these biomarkers in eggs of commercially farmed pikeperch (44 females). We measured egg TAC, as well as lesion rates per 10 kb of 12S and cytochrome b (cytb) as target regions within the mitochondrial genome by qPCR. It was tested whether these markers correlate with embryo development (fertilization rate, embryo survival, hatching rate). There was no significant relation of mtDNA lesion rates or TAC with these egg quality parameters. We detected average lesion rates (± SD) of 1.50 (± 1.57) and 1.89 (± 2.14) in 12S and cytb mtDNA respectively. Lesion rates in 12S and cytb were highly correlated within samples (P < 0.0001) and were independent of the observed TAC. The results suggest that TAC does not prevent mtDNA fragmentation and that embryos rather seem to be able to cope with the observed fragmentation of mtDNA. However, in post-ovulatory aged eggs of three females with little to no fertilization success, lesion rates of cytb were significantly elevated, whereas TAC was significantly lower compared to other females, suggesting a possible role of oxidative stress during post-ovulatory ageing.

Some quantitative indicators of postovulatory aging and its effect on larval and juvenile development of Atlantic salmon (Salmo salar).

Modern out-of-season egg production in Atlantic salmon (Salmo salar) increases the risk of postovulatory aging (POA) of oocytes. Postovulatory aging is known to influence oocyte quality in salmonids, but reliable tests for POA are lacking in Atlantic salmon egg production. To address this problem, we have collected oocytes from the same 20 Atlantic salmon females sequentially in approximately 1-week intervals, from the start of ovulation until 28 days postovulation (dpo), to determine the effect of natural retention of matured oocytes in body coelomic cavity on further performance of embryos and juveniles produced from those oocytes. Also, we investigated oocyte water hardening and several coelomic fluid parameters as potential quantitative indicators of POA. Oocyte quality decreased significantly from 22 dpo onward, as inferred from decrease in fertilization success and survival of embryos, alevins, and juveniles and increase in alevin and juvenile deformity rates. The occurrence of head deformities was significantly related to postovulatory age of oocytes. Coelomic fluid pH decreased significantly at 28 dpo and correlated positively with fertilization rates (r = 0.45), normal eyed embryo rates (r = 0.67), and alevin relative survival rates (r = 0.63) and negatively correlated with total alevin deformity rates (r = -0.59). Oocyte weight gain at 60 minutes decreased significantly at 28 dpo and correlated negatively with total alevin deformities and the occurrence of cranial nodules (r = -0.99). Generally, quality of ovulated oocytes remained stable for the first 2 weeks after ovulation. Later on, POA negatively influenced Atlantic salmon embryo, alevin, and juvenile performance. For the first time, we show a long-term effect of POA on salmonid juvenile performance. Standardized pH measurements of coelomic fluid could potentially improve embryo and juvenile production by identifying low-quality oocytes at an early stage during the production.

Cambios morfológicos en gametos del barbo tigre Puntius tetrazona (Cypriniformes: Cyprinidae) e implementación de la fertilización in vitro / Morphological changes in gametes of tiger barb Puntius tetrazona (Cypriniformes: Cyprinidae) and the implementation of in vitro fertilization

The production of ornamental fishes represents an economic activity of a growing number of Mexican families. Nevertheless, the reproduction of fish in captivity is one of the complications faced by farmers. This study was set up to: (i) evaluate the morphological and functional changes induced by hydration in the gametes of fish tiger barb (Puntius tetrazona; 240 samples) at tree times after hydration (10, 20 and 30s) with classic spermograms (volume, sperm concentration, viability, motility, and normal morphology); and (ii) evaluate the implementation of in vitro fertilization based on the ovulation rate, the percentage of fertilization and hatching, and the larval numbers obtained after 72 hours. The average volume of milt was 3.0±0.7μL, and the minimum, maximum and average concentration of sperm was 44.4x10(6) spz/mL, 52.3x10(6)spz/mL, and 48.1±5.9x10(6)spz/mL, respectively. The viability and motility of the sperm was 84.6±3.2% and 81.5±2.2%, respectively. The diameter of the sperm with/without water contact was 2.1±0.6μm and 3.8±1.0μm (p<0.05); the largest diameter was recorded 30 seconds after the contact with water. For oocytes, the smaller and larger diameters were recorded at 10 and 30s, respectively (both with/without water contact); the oocytes diameters after 10 and 30 seconds of contact with water were 1.11 and 1.55mm, respectively. A higher ovulation rate was recorded using the in vitro fertilization: 250±50 oocytes versus 28±09 oocytes (during natural fertilization; p<0.05). Nevertheless, fertilization and hatching rates were higher for the natural fertilization (80 and 60%, respectively). Considering the number of larvae obtained after 72 hours, our results showed a higher value for the in vitro fertilization (75±18 compared to 13.4±12 of the natural fertilization; p<0.05). We propose this fish as a model for other ornamental fishes of commercial interest. Our results demonstrate that the in vitro fertilization is a very high viable option to optimize and maximize resources; besides, the reproduction management optimization under controlled conditions may enhance wild fish stocks preservation. Rev. Biol. Trop. 62 (4): 1353-1363. Epub 2014 December 01.

El objetivo del presente estudio fue conocer las características de los gametos de Puntius tetrazona (n=240), los cambios morfológicos a partir de su activación mediante espermogramas cásicos y por otro lado, se evaluó la implementación de la fertilización in vitro a partir de la tasa ovulatoria, el % de fertilización y eclosión y el número de larvas vivas a las 72h. El volumen promedio de semen fue de 3.0±0.7µL. La concentración espermática mínima, máxima y promedio fue 44.48x10(6)spz/mL, 52.3x10(6)spz/mL y 48.1±5.9x10(6)spz/mL, respectivamente. La viabilidad promedio fue de 84.68±3.27%. La motilidad promedio fue 81.53±2.28%. El diámetro de los espermatozoides fluctuó entre 2.16±0.2 y 2.79±0.3µm; 3.84±0.3 y 4.86±0.31µm sin y con contacto con el agua respectivamente, con diferencias significativas. El diámetro mayor fue a los 30s en contacto con el agua. Los ovocitos de menor y mayor diámetro se registraron a los diez y 30s sin y con contacto con el agua respectivamente. Los diámetros de los ovocitos en diez y 30s en contacto con el agua fluctuaron entre 1.11 y 1.55mm respectivamente. La mayor tasa ovulatoria fue en la fertilización in vitro con 250±50 ovocitos frente a 28±09 de la natural, con diferencias significativas. Los porcentajes de fertilización y eclosión fueron más elevados en la fertilización natural con 80% y 60% respectivamente. Se registraron 75±18 larvas a las 72 horas en el grupo in vitro comparado con 13.4±12 larvas de la fertilización natural. Con lo anterior, la técnica que permitió mayor cantidad de larvas fue la de fertilización in vitro.