In vivo and in vitro aging of common carp Cyprinus carpio sperm after multiple hormonal application and stripping of males.

The present study was designed to evaluate sperm phenotypic variables during in vivo and in vitro storage following multiple sperm stripping in common carp (Cyprinus carpio L.). Each male was injected 3 times with carp pituitary 3 days apart. Sperm was stored in vivo in the body cavity for 0.5 days (Fresh sperm) and 3 days (Old sperm) after hormonal stimulation. Then sperm was collected and diluted with a carp extender at a ratio of 1:1, and stored in vitro on ice for 0, 3, and 6 days. The phenotypic parameters, including the number of total motile spermatozoa, number of fast motile spermatozoa, number of motile spermatozoa, percentage of fast motile spermatozoa, and percentage of spermatozoa motility were the major components of principal component analysis (PCA). In general, Fresh sperm from the first stripping showed slightly better quality than Old sperm from the second and third stripping, especially in the phenotypic parameters of a number of total spermatozoa and a number of total motile spermatozoa (P < 0.05). The highest kinetic and quantitative spermatozoa variables were obtained in Fresh and Old sperm just after sperm collection (0-day storage in vitro), and then they were decreased during the period of in vitro storage up to 6 days (P < 0.05). However, the fertilization, hatching, and malformation rates from Fresh sperm were similar compared with the Old sperm. Sperm could be stripped 0.5 days post hormonal treatment and stored in vitro up to 6 days with good fertilization performance (fertility, hatching, and malformation rates were 92.5%, 91.5%, and 1.3%, respectively). Therefore, our results suggested that multiple hormonal treatments with multiple stripping could be used in artificial reproduction in common carp.

Common carp (Cyprinus carpio) sperm reduction during short-term in vitro storage at 4 °C.

This study was designed to optimize a short-term storage protocol for common carp sperm at 4 °C under aerobic condition. Sperm from individual males were collected directly with or without extenders. The results demonstrated that in general, it was similar effect to collect sperm directly in extenders and keeping sperm for 0.5 h after collection without extenders. Sperm was diluted with eight selected extenders (sperm: extender = 2:1, 1:1 and 1:9) and undiluted sperm was used as a control. Sperm and seminal plasma parameters (sperm motility, velocity, membrane integrity, sperm concentration, osmolality and pH in seminal plasma) were evaluated in sperm stored on ice under aerobic conditions at 0, 2, 4, 6 and 8 days post stripping (DPS) using the computer- assisted sperm analysis system. Results showed that 1:1 and 2:1 dilution maintained higher sperm function and more sperm for a longer period. After 8 DPS, the best sperm quality and quantity was recorded in the common carp seminal plasma supplemented with 50 mM NaCl, Cejko extender (2 mM CaCl2, 1 mM MgSO4, 20 mM Tris, 110 mM NaCl, 40 mM KCl, pH 7.5 and 310 mOsm/kg) supplemented with/without 25 mM KCl/NaCl. The reduction of spermatozoa number with time during short-term storage but varied according to different dilution ratios and extenders. At 8 DPS, sperm count has dropped by 22.9 % in a dilution of 1:1 compared to 50.3 % in sperm without dilution. Extenders with diluted 1:1, especially Cejko solution, largely postponed sperm reduction, 21.3 % compared to 55.5 % for sperm stored without extenders.

Optimization of Sperm Management and Fertilization in Zebrafish (Danio rerio (Hamilton)).

The aim of the present study was to investigate the spontaneous motility of spermatozoa and to optimize sperm collection, short-term sperm storage, and fertilization in zebrafish Danio rerio. The movement of spermatozoon in water was propagated along the flagellum at 16 s after sperm activation then damped from the end of the flagellum for 35 s and fully disappeared at 61 s after activation. For artificial fertilization, milt must be added to an immobilizing solution, which stops the movement of sperm and keeps the sperm motionless until fertilization. E400 and Kurokura as isotonic solutions were shown to be suitable extenders to store sperm for fertilization for 6 h. E400 stored sperm for 12 h at 0-2 °C. Sperm motility decreased only to 36% at 12 h post stripping for the E400 extender and to 19% for the Kurokura extender. To achieve an optimal level of fertilization and swim-up larvae rates, a test tube with a well-defined amount of 6,000,000 spermatozoa in E400 extender per 100 eggs and 100 µL of activation solution has proven to be more successful than using a Petri dish. The highest fertilization and swim-up larvae rates reached 80% and 40-60%, respectively, with milt stored for 1.5 h in the E400 extender at 0-2 °C.

Changes in Phenotypes and DNA Methylation of In Vitro Aging Sperm in Common Carp Cyprinus carpio.

The purpose of the current study was to analyze phenotypic and functional characteristics of common carp (Cyprinus carpio) spermatozoa during in vitro aging and to investigate whether global DNA methylation is affected by sperm aging. Milt was collected from five individual males, stored in vitro on ice in a refrigerator for up to 96 h post stripping (HPS) and used to fertilize eggs with intervals of 1, 24 and 96 h. Computer-assisted sperm analysis and a S3e Cell Sorter was employed to determine the spermatozoa phenotypic characteristics (motility, velocity, concentration and viability). In addition, pH and osmolality of the seminal fluid and the capacity of the spermatozoa to fertilize, hatching rate and health of the resulting embryos were examined at different aging times. Whole-genome bisulfite sequencing was used to compare the global and gene-specific DNA methylation in fresh and aged spermatozoa. The results demonstrated that spermatozoa aging in common carp significantly affects their performance and thus the success of artificial fertilization. The methylation level at the cytosine-phosphate-guanine (CpG) sites increased significantly with 24 HPS spermatozoa compared to the fresh group at 1 HPS and then decreased significantly at 96 HPS. A more detailed investigation of gene specific differences in the DNA methylation was hindered by incomplete annotation of the C. carpio genome in the public databases.

Estradiol Upregulates the Expression of the TGF-ß Receptors ALK5 and BMPR2 during the Gonadal Development of Schizothorax prenanti.

TGF-ß receptors play important roles in mediating TGF-ß signals during gonadal development. To identify the functions of TGF-ß receptors, including the type I receptor (activin receptor-like kinase 5, ALK5) and type II receptor (bone morphogenetic protein receptor 2, BMPR2), during the gonadal development of S. prenanti, the full-length cDNA sequences of ALK5 and BMPR2 were isolated and characterized. Their expression patterns in developing gonads and in the gonads of exogenous estradiol (E2) -fed fish were analyzed. The cDNAs of ALK5 and BMPR2 were 1925 bp and 3704 bp in length and encoded 501 and 1070 amino acid residues, respectively. ALK5 and BMPR2 were mostly expressed in gonads, particularly in cortical alveoli stage ovaries and mid-spermatogenic stage testes; however, the overall level of BMPR2 mRNA was higher than that of ALK5 during gonadal development. Furthermore, immunohistochemical signals of ALK5 and BMPR2 were mostly detected at chromatin nucleolar oocytes and perinuclear oocytes in ovaries and at spermatocytes and spermatogonia in testes. Exogenous E2 induces the gonadal expression of ALK5 and BMPR2, and BMPR2 is more responsive to E2 than ALK5. These results suggest that ALK5 and BMPR2 might play a potentially vital role in both folliculogenesis and spermatogenesis in S. prenanti.

Expression patterns and oestradiol regulation of growth differentiation factor 9 in Schizothorax prenanti.

Growth differentiation factor 9 (GDF9) plays pivotal roles in regulating follicular development in many mammalian species. In the present study, the full-length Schizothorax prenanti gdf9 cDNA sequence was isolated and characterized, and its expression pattern in developing gonads and in the gonads of exogenous oestradiol (E2)-fed fish were analysed. The S. prenanti gdf9 cDNA sequence consisted of 1958 base pairs (bp), encoded a 413 amino acid, and showed high sequence similarity with Carassius gibelio and Cyprinus carpio. The quantitative real-time PCR analysis revealed that gdf9 was mainly expressed in the gonads, with particularly high expression in the cortical alveoli stage ovary and late-spermatogenic stage testis. Immunohistochemical signals for Gdf9 were mainly detected in chromatin nucleolar oocytes, spermatogonia and spermatocytes. Furthermore, the gonadal expression of gdf9 induced by exogenous E2 was related to the feeding time and dose. Taken together, these findings were helpful to gain a better understanding of the role of Gdf9 in both folliculogenesis and spermatogenesis in S. prenanti.

Characterization and expression profiles of cyp19a1a in the schizothoracine fish Schizothorax prenanti.

Aromatase plays a central role in ovarian differentiation and development in teleosts. In the present study, we identified a cyp19a1a homologue from the ovary of Schizothorax prenanti and analysed its expression at both the mRNA and protein levels. Cyp19a1a of S. prenanti showed high homology with that of other teleosts, especially S. kozlovi. The ovary and testis were the main sites of cyp19a1a expression in S. prenanti, and cyp19a1a transcript levels peaked in the mid-vitellogenic (MVG)-stage ovary and the mid-spermatogenic (MS)-stage testis. Signals of Cyp19a1a immunopositivity were detected in the spermatocytes and follicular cells of cortical alveolar-stage and MVG oocytes but not in spermatogonia or spermatids. Taken together, these findings indicate that Cyp19a1a may play an important role in oocyte vitellogenesis as well as spermatocyte development in S. prenanti.

The behavior of umami components in thermally treated yeast extract.

Umami proteolytics are natural food flavor alternatives to glutamate. In this study, key umami taste fractions were separated and purified from thermally treated yeast extract (YE) to yield fifteen umami peptides. Systematic approaches using sensory-guided fractionation on taste-active umami proteolytics separation and detection were utilized. A reaction temperature of 110 °C was optimum for umami peptide generation. Under this reaction temperature, the sensory score and E-tongue results of umami taste were the highest. The sensory evaluation-based taste dilution analysis and taste threshold determination supported the hypothesis that umami peptides have their physiological effect by binding to G-protein coupled receptors. The structural differences of umami peptides contribute to their taste profile and allow categorization into two group Types. Fifteen umami peptides were then categorized into Type I and Type II regarding the contractual-based taste mechanism: Type I peptides imparted complex tastes. The tastes of Type I peptides could split into two stages: bitterness and umami in pure water, whereas, Type II peptides presented strong umami taste at a high concentration in pure water, and the relationship between umami capacity and peptides concentration was linear. Finally, the guidance of the umami peptide usage in the flavor industry has been established according to broths dissolution test.

Changes in the perception of bitter constituents in thermally treated yeast extract.

BACKGROUND: Many studies have been performed over the past four decades to identify and quantify the odor-active key volatiles in yeast extract (YE) but knowledge of the nonvolatile taste compounds is still rather fragmentary. In particular, research on bitter peptides with various structures during the thermal treatment of YE is still scarce. RESULTS: Compounds imparting a bitter taste to thermally treated YE were investigated using sensory-guided fractionation. This research found that when the treatment temperature reached 130 °C, bitter peptides were generated. Sensory evaluation of the purified, synthesized peptides revealed that four of these peptides showed a pronounced bitter taste with a taste dilution (TD) factor from 5 to 9. Guidance is provided for the production of bitter peptides in the flavor industry. CONCLUSION: Based on results from previous work on umami peptides, and this study, keeping the thermal reaction temperature under 120 °C could maximize the umami flavor and control bitterness so that it remains in an acceptable range. © 2019 Society of Chemical Industry.

Flavour-active compounds in thermally treated yeast extracts.

BACKGROUND: Aroma-active compounds and non-volatile substances determine the characteristic aroma and taste of yeast extract (YE). Changes in the characteristic aroma and taste of YE due to thermal reaction are rarely studied, and the relationship between aroma-active compounds and non-volatile compounds is not yet clear. RESULTS: Non-volatile compounds identified by HPLC and LC/MS/MS were reduced by a rise in temperature, except for some amino acids. Peptides underwent degradation. In addition, a further rise in temperature above 120 °C resulted in a bitter and sour taste. Furans, pyrazines, thiophenes, thiazoles and some branched chain sulfur compounds were derived from GC/O/MS (SPME and SAFE). Sensory results revealed that the concentration of volatile compounds increased with an increase in temperature. The overall aroma profiles of YE at 25, 100 and 110 °C were buttery, green, nutty and meaty, while YE at 140 °C had a strong sour and sulfur odour. CONCLUSION: The non-volatile compounds of YE were reduced and different volatile compounds were produced under different thermal treatments. There was a negative correlation between these two types of compounds. The different taste sensors and all precursors were correlated with each other. There are significant relationships between different odorants and aroma-active compounds of YE after thermal treatment. © 2018 Society of Chemical Industry.

The complete mitochondrial DNA of Ancherythroculter wangi.

The complete mitochondrial genome of Ancherythroculter wangi was determined. The mitochondrial genome, consisting of 16,622 base pairs (bp), encoded 13 protein-coding genes, 2 ribosomal RNAs, 22 transfer RNAs and a non-coding control region, as those found in other Ancherythroculter species. These results can provide useful data for further studies on taxonomic status, molecular systematics, and stock evaluation.

[Research of spectrum characteristics for light conversion agricultural films].

The solar spectrum and the function spectrum in chrysanthemum and tomato were determined in this paper. The research for a relation plant growth to solar spectrum showed that the efficiency of plant making use of ultraviolet light of 280-380 nm and yellow-green light of 500-600 nm and near IR spectra over 720 nm are lower, that the blue-purple light of 430-480 nm and red light of 630-690 nm are beneficial to enhancing photosynthesis and promoting plant growth. According to plant photosynthesis and solar spectrum characteristic, the author developed CaS:Cu+, Cl- blue light film, and red light film added with CaS:Eu2+, Mn2+, Cl- to convert green light into red light, and discussed the spectrum characteristic of red-blue double peak in agricultural film and rare earth organic complex which could convert ultraviolet light into red light. Just now, the study on light conversion regents in farm films is going to face new breakthrough and the technology of anti-stocks displacement to study red film which can convert near infrared light are worth to attention.