Metabolite profiling of the post-ovulatory oocytes of the common carp, Cyprinus carpio: A 1H NMR-based metabolomics approach.

A metabolomics study was conducted to investigate the molecular bases of oocyte over-ripening in common carp, Cyprinus carpio from a metabolic point of view. The ovulation was induced in fish brooders by intramuscular injection of pituitary extract and oocytes were collected four times post-ovulation with 30 min intervals. A set of 32 metabolites were identified on the NMR spectra of the oocytes, which mainly included energy-linked metabolites, amino acids, methylated metabolites and citric acid cycle (TCA) intermediates. PCA and PLS-DA models clearly separated the post ovulations times, indicating the effects of post-ovulation time on oocyte metabolome content. Based on the loading plot outputs, 15 metabolites including tryptophan, cysteine, AMP, tyrosine, valine, creatine phosphate (PCr), ATP, leucine, inosine, malate, acetate, TMAO, glucose, fumarate and lysine had more effects on the separation of post ovulation times. According to the results of metabolite profiling, the concentrations of glutamine, alanine, tryptophan, lysine and cysteine mostly significantly (P < 0.01) increased at 90 and 120 min post-ovulation. The concentrations of PCr, ATP, inosine and guanosine were relatively stable until 60 min post-ovulation, while significantly (P < 0.01) decreased at 90 and 120 min post ovulation. The TCA metabolites succinate, malate and fumarate significantly (P < 0.01) elevated at 90 and 120 min post-ovulation. AMP concentrations remained relatively unchanged until 30 min and then progressively decreased with time post ovulation (P < 0.01). The concentrations of lactate showed significant elevations at 90 and 120 min post ovulation (P < 0.01). In conclusion, the energetic potentials of the oocytes reduced with time post ovulation. There were apparent elevations in the concentrations of free amino acids, which may be associated with the onset of proteolytic activities in the post ovulatory oocytes. In addition, we found some changes in the apoptotic-related metabolites, which may support the results of previous studies regarding the oxidative stress and following apoptosis in post ovulatory oocytes of fish.

Size-dependent effects of microplastic on uptake, immune system, related gene expression and histopathology of goldfish (Carassius auratus).

Todays, with the industrialization of human societies, pollution of aquatic ecosystems with plastics derivatives are a serious concern, affecting the life of their organisms. The present study was conducted to investigate the size effects of micro-plastic, polystyrene on some physiological lesions of the goldfish, Carassius auratus. Fish were exposed to two sizes (0.25 and 8 µm) polystyrene at different environmentally relevant concentrations. The exposure trial was done in two steps. First, fish exposed to a stable concentration of 300 mg/L polystyrene for 168 h. Gill, intestine, and liver tissues were sampled every 24 h to investigate the accumulation of polystyrene. Then, fish were exposed in three replicates to 0 (control), 0.05, 0.5, and 5 mg/L polystyrene in two sizes of 0.25 and 8 µm for 28 days. After the exposure period, gill, liver, and intestine tissues were sampled for histological study, also, serum samples were collected for biochemical assays. Fluorescent microscope observations confirmed the accumulation of polystyrene in tissue samples with time. In addition, histological lesions were found in the liver, intestine, and gill of the exposed fish. The severity of lesions showed a size and dose-dependent pattern. Polystyrene induced the antioxidant system of exposed fish through elevating the levels of SOD and CAT activity and significant difference in expression of antioxidant related genes (CAT, SOD and HSP70). In conclusion, the results of the present study confirmed the toxic effects of microplastic, polystyrene on goldfish.

Effect of oral administration of GnRHa+nanoparticles of chitosan in oogenesis acceleration of goldfish Carassius auratus.

Several methods have been used to accelerate previtellogenesis and vitellogenesis stages in fish, including hormonal induction, sustained-release delivery systems, and oral delivery of gonadotropin-releasing hormone (GnRH). In this study, we proposed the oral administration of GnRH analog + nanoparticles of chitosan to accelerate oogenesis in goldfish as a model fish in reproductive biology and aquaculture. In this regard, adult female goldfish were fed with six experimental groups: chitosan, 50 µg GnRHa/kg b.w., 100 µg GnRHa/kg b.w., chitosan + 50 µg GnRHa/kg b.w., and chitosan + 100 µg GnRHa/kg b.w., and diet without any additive as the control for 40 days in triplicate. Every 10 days, ovarian samples were collected, and gonadosomatic index (GSI), oocyte diameter (OD), zona radiata thickness (Zr), and diameter of the follicular layer (Fl) were measured to assess ovarian developmental stage for each treatment. Additionally, blood sampling was done to measure serum 17ß-estradiol concentration at the end of the experiment. All parameters remained unchanged during the experiment in the chitosan-fed group. In the group fed with 100 µg GnRH or chitosan nanoparticle + 100 µg GnRHa, these parameters in general were increased. However, the effects in 50 µg GnRHa or chitosan nanoparticle + 50 µg GnRHa treatments were uncertain; they affected serum E2 levels as a trend toward a significant increase was observed in goldfish treated with chitosan nanoparticle + 100 µg GnRHa. Finally, the results indicated the oral administration of chitosan + 100 µg GnRHa/kg b.w. significantly accelerated the oocyte development and growth of ovary.

Droplet vitrification versus straw cryopreservation for spermatozoa banking in Persian sturgeon (Acipenser persicus) from metabolite point of view.

Persian sturgeon (Acipenser persicus), a commercially valuable and critically endangered fish species has been suffering considerable declines in populations in the nature due to over-fishing, habitat destruction and marine pollution during past decades. Since there were no achievements in artificial reproduction programs, genetic resource banking such as gametes and embryo cryopreservation can be a good strategy however, reported resulting gamete qualities were considerably low. In the present study, the metabolome content of Persian sturgeon spermatozoa was investigated during common straw cryopreservation and novel droplet vitrification by the use of 1H NMR (Nuclear Magnetic Resonance) spectroscopy. Univariate (ANOVA) and multivariate (PCA) analysis showed significant differences in the metabolic profiles between cryopreserved and fresh spermatozoa samples. Adenine, creatine, creatine phosphate, glucose, guanidoacetate, lactate, N, N-dimethylglycine, and glycine levels showed no significant differences between these two cryopreservation techniques suggesting these metabolites and their corresponding enzymes and chemical pathways are so vulnerable to the temperature changes and even higher cooling rate in droplet vitrification could not conserve them. However, significant differences were found in acetate, creatinine, betaine, ß-alanine and trimethylamine N-oxide suggesting better efficiency of droplet vitrification in protection of some metabolites associated to spermatozoa energetics, redox balance and hypoxia compensation compared to straw cryopreservation.

1H NMR metabolic profiling of the cryopreserved spermatozoa of the wild endangered Persian sturgeon (Acipenser persicus) with the use of beta-cyclodextrin as an external cryoprotectant.

Persian sturgeon (Acipenser persicus) is an endangered species and genetic resource banking such as gametes and embryo preservation could be one of the most pursued conservation approaches. In this study, deleterious effects of the traditional cryopreservation technique and the effect of different doses of 2-hydroxypropyl-beta-cyclodextrin (HßCD) on thawed spermatozoa quality (motility duration and percentage) of Persian sturgeon (Acipenser persicus) were investigated from metabolic aspects of view. For cryopreserving, semen was diluted with Tris-HCl (100 mM) extenders containing 0, 5, 10, and 15 mM of HßCD in a ratio of 1:1 (semen/extenders). Semen-extenders were filled into 0.5-mL straws and were frozen with the vapor of liquid nitrogen, and then immersed into liquid nitrogen. Cryopreserved spermatozoa were thawed in water baths in 15 s. Two treatments with the highest and the lowest motility percentages (0 and 10 mM of HßCD) were chosen to reveal the extremes of the metabolites change range and were objected to 1H NMR spectroscopy. Univariate (ANOVA) and multivariate (PCA) analysis of the obtained metabolic profiles showed significant changes (P < 0.05) in metabolites. The use of 10 mM of HßCD was completely successful in the preservation of creatinine, glucose, guanidoacetate, O-phosphocholine, and N, N-dimethylglycine and probably their corresponding biochemical pathways, but it failed to preserve lactate, carnitine, betain, ß-alanin, and trimethylamine N-oxide. It was also partially successful in preserving acetate, creatine, creatine phosphate, and glycine, all suggesting how HßCD can be effective as a cryoprotectant.

Metabolic changes in droplet vitrified semen of wild endangered Persian sturgeon Acipenser persicus (Borodin, 1997).

Comparative quantitative metabolite profiling can be used for better understanding of cell functions and dysfunctions in particular circumstances such as sperm banking which is an important approach for cryopreservation of endangered species. Cryopreservation techniques have some deleterious effects on spermatozoa which put the obtained results in controversy. Therefore, in the present study, quantitative 1H NMR (Nuclear Magnetic Resonance) based metabolite profiling was conducted to evaluate metabolite changes related to energetics and some other detected metabolites in vitrified semen of critically endangered wild Acipenser persicus. The semen was diluted with extenders containing 0, 5, 10, and 15 µM of fish antifreeze protein (AFP) type III as a cryoprotectant. Semen-extenders were vitrified and stored for two days. Based on post-thaw motility duration and motility percentage assessments, two treatments with 10 µM and 0 µM of AFP had the highest and the lowest motility percentages respectively and they were objected to 1H NMR spectroscopy investigations in order to reveal the extremes of the metabolites dynamic range. Univariate (ANOVA) and multivariate (PCA) analysis of the resulting metabolic profiles indicated significant changes (P > 0.05) in metabolites. The level of some metabolites including acetate, adenine, creatine, creatine phosphate, lactate, betaine, sarcosine, ß-alanine and trimethylamine N-oxide significantly decreased in vitrified semen while some others such as creatinine, guanidinoacetate, N, N-dimethylglycine, and glycine significantly increased. There were also significant differences between vitrified treatments in levels of creatine, creatine phosphate, creatinine, glucose, guanidinoacetate, lactate, N, N-dimethylglycine, and glycine, suggesting how fish AFP type III can be effective as a cryoprotectant.