Cryopreservation of sperm from the gudgeon, Microphysogobio rapidus (Cyprinidae): Effects of cryoprotectant, diluents, and dilution ratio.

We investigated methods for cryopreserving sperm from the endangered gudgeon, Microphysogobio rapidus, by examining the effects of cryoprotective agent (CPA) concentration, diluent, and dilution ratio on post-thaw sperm quality. The quality of frozen sperm was evaluated in terms of motility and kinematic parameters, viability, DNA damage, and fertilization rate. We evaluated methanol, glycerol, dimethyl sulfoxide (DMSO), and ethylene glycol as CPAs. Sperm motility, velocity, and viability were significantly higher when methanol was used as the CPA (p < 0.05). The diluents tested were Ringer's solution, Kurokura's Extender, Common Carp Sperm Extender (CCSE), and buffered sperm motility-inhibiting saline solution (BSMIS); post-thaw motility was highest when Ringer's solution was used as the diluent. Next, various quantities of methanol were combined with Ringer's solution to identify the optimal dose of methanol. The dilution ratios tested ranged from 1:1 to 1:7. Cryopreserved sperm was thawed at 20 °C for 15 s. The use of 10% methanol with Ringer's solution at a dilution ratio of 1:5 resulted in the highest post-thaw sperm motility, viability, and velocity including VAP, VCL, and VSL. Post-thaw sperm showed significantly greater DNA damage than the control (fresh sperm) (p < 0.05). The fertilization rate was highest with fresh sperm (p < 0.05), followed by sperm frozen with 10% methanol + Ringer's solution. We recommend that the best way to preserve sperm in the studied species is to use a combination of Ringer's solution and 10% methanol at a 1:5 dilution ratio. Our findings will facilitate the artificial fertilization of M. rapidus.

Purification of phenoloxidase from Haliotis discus hannai and its anti-inflammatory activity in vitro.

Haliotis discus hannai, a food with a high protein content, is widely consumed in Asian countries. It is known to have antioxidant, anticancer, and antibacterial effects. Since the biological significance of H. discus hannai hemolymph has not been widely studied, the objective of the present study was to purify phenoloxidase (PO) and investigate its immunological effects on human colonic epithelial cells. PO was purified through ammonium sulfate precipitation and one step column chromatography. The molecular weight of the protein was about 270 kDa. When PO was mixed with Gram-negative bacteria-derived lipopolysaccharide (LPS) at various ratios (10:1-1:10, w/w), the amount of residual LPS was reduced. PO at concentrations up to 200 µg/mL was not cytotoxic to HT-29 cells. The inflammatory response induced by LPS in HT-29 cells was regulated when the concentration of PO was increased. With increasing concentration of PO, production levels of pro-inflammatory cytokines, cytokines associated with hyperimmune responses such as IL4, IL-5, and INF-γ, and prostaglandin 2 (PGE2) were regulated. It was thought that simultaneous treatment with PO and LPS anti-inflammatory effects in HT-29 cells showed by regulating the ERK1/2-mediated NF-κB pathway. Results of this study suggest that H. discus hannai hemolymph is involved in the regulation of Gram-negative bacteria-related inflammatory immune responses in human colonic epithelial cells.

Intermediate-Term Storage of Spotted Halibut (Verasper variegatus) Sperm: Effects of Storage Methods, Extenders Supplemented with Antibiotics and Antioxidants on Sperm Quality.

Intermediate-term preservation of sperm assists the reproductive management of fish spermatozoa; however, no information is available on sperm of the spotted halibut, Verasper variegatus. We aimed to identify the optimum diluents, temperatures, dilution ratios, antibiotics, and antioxidants for sperm motility and cell viability. The diluents evaluated were marine fish Ringer's solution (MFRS), Stein's solution, 300 mM sucrose, and 300 mM glucose (diluted 1:1 [sperm: diluent], 1:2, 1:4, and 1:10 and stored at 0, 2, 4, and 6 °C). Neomycin and gentamycin (100, 200, 400, and 800 mg/L) and antioxidants (Mito-TEMPO [0, 25, 50, 75, 100, 125, 150, 175, and 200 µM], reduced glutathione [0, 2, 4, 6, 8, and 10 mM], and trehalose [0, 50, 100, 150, 200, and 250 mM]) were assessed in terms of sperm preservation. The most effective condition for cold storage of spotted halibut sperm was Stein's solution at a dilution ratio of 1:4 at 2 °C, with a combination of neomycin 800 mg/L and 250 mM trehalose that showed spermatozoa motility of > 43% after 60 days. These storage conditions will be valuable for spotted halibut hatcheries.

Cryopreservation of Roughscale Sole (Clidoderma asperrimum) Sperm: Effects of Cryoprotectant, Diluent, Dilution Ratio, and Thawing Temperature.

The roughscale sole, Clidoderma asperrimum is categorized as an endangered species. Sperm freezing is essential for preserving gametes. This study examined the CPA concentration, diluent, dilution ratio, and thawing temperature to design a sperm cryopreservation protocol for roughscale sole. The variables examined included sperm motility and kinematics, cell survival, fertilization, and DNA fragmentation. Sperm motility parameters were assessed via computer-assisted sperm analysis using a CEROS II instrument. Cell survival rate and DNA damage were assessed using the Cell Counting Kit-8 and single-cell gel electrophoresis assay, respectively. Sperm preservation was tested using several CPAs, including ethylene glycol, dimethyl sulfoxide (DMSO), glycerol, propylene glycol, and methanol. The diluents tested were 300 mM sucrose, 300 mM glucose, Stein's solution, Ringer's solution, and Hank's solution. The optimal conditions for sperm cryopreservation were 10% DMSO + Stein's solution. After thawing, sperm motility was highest with a 1:1 dilution ratio (sperm to CPA + diluent), at 69.20 ± 0.32%; thawing at 10 °C was optimal for post-thaw motility (72.03 ± 0.95%). The highest fertilization rate (40.00 ± 1.22%) was obtained using DMSO. The fresh sperm had the lowest tail DNA, followed by 10% DMSO + Stein's solution. The developed cryopreservation methods can be used in roughscale sole hatcheries.

Characterization of a C-Type Lectin Domain-Containing Protein with Antibacterial Activity from Pacific Abalone (Haliotis discus hannai).

Genes that influence the growth of Pacific abalone (Haliotis discus hannai) may improve the productivity of the aquaculture industry. Previous research demonstrated that the differential expression of a gene encoding a C-type lectin domain-containing protein (CTLD) was associated with a faster growth in Pacific abalone. We analyzed this gene and identified an open reading frame that consisted of 145 amino acids. The sequence showed a significant homology to other genes that encode CTLDs in the genus Haliotis. Expression profiling analysis at different developmental stages and from various tissues showed that the gene was first expressed at approximately 50 days after fertilization (shell length of 2.47 ± 0.13 mm). In adult Pacific abalone, the gene was strongly expressed in the epipodium, gill, and mantle. Recombinant Pacific abalone CTLD purified from Escherichia coli exhibited antimicrobial activity against several Gram-positive bacteria (Bacillus subtilis, Streptococcus iniae, and Lactococcus garvieae) and Gram-negative bacteria (Vibrio alginolyticus and Vibrio harveyi). We also performed bacterial agglutination assays in the presence of Ca2+, as well as bacterial binding assays in the presence of the detergent dodecyl maltoside. Incubation with E. coli and B. subtilis cells suggested that the CTLD stimulated Ca2+-dependent bacterial agglutination. Our results suggest that this novel Pacific abalone CTLD is important for the pathogen recognition in the gastropod host defense mechanism.

Heat shock protein 70 gene expression and stress response of red-spotted (Epinephelus akaara) and hybrid (E. akaara female × E. lanceolatus male) groupers to heat and cold shock exposure.

The importance of the temperature tolerance of fish is increasing due to climate change caused by global warming. This study examined the expression of the heat shock protein 70 (HSP70) gene, and plasma cortisol and glucose levels, as a stress response in red-spotted and hybrid groupers during exposure to heat and cold shock. Temperature in the tank where fishes acclimated at 20℃ was gradually increased or decreased, respectively, to examine the survival rate of fish. The result showed a higher survival rate of the hybrid than that of the red-spotted grouper upon exposure to a higher temperature. To further analyze the factors associated with temperature-associated stress, fishes were collected from different temperatures which changed from 20 to 30℃ or 10℃, respectively, and then back to 20℃. The expression levels of the gene encoding heat shock protein 70 (HSP70) were analyzed by qPCR using cDNA prepared from RNA extracted from the brain. A higher level of HSP70 transcript was detected in the hybrid during heat shock exposure. Analysis of cortisol and glucose from the blood of fish collected during the acclimation periods clearly indicated that the level of cortisol was increased upon temperature shift although a slight difference in the degrees of changes timing was slightly different between red-spotted grouper and hybrid. The results showed a correlation between the level of HSP70 and survival rate upon exposure to higher temperature shock. This study provides basic information regarding whether HSP70 expression increases the survival rate of fish subjected to rapid temperature changes.

Dead-End (dnd) Gene Cloning and Gonad-Specific Expression Pattern in Starry Flounder (Platichthys stellatus).

dnd is a germline-specific maternal RNA expressed in various vertebrate classes, which encodes an RNA-binding protein that is essential for PGC migration. The purpose of this study is fundamental research about starry flounder dnd gene for germ cell marker development. In this study, we cloned and analyzed the expression levels of Platichthys stellatus dead end (psdnd) in various tissues and embryonic stages. The psdnd gene was isolated from starry flounder ovaries, cloned into a pGEM-t vector, and sequenced. Full-length of psdnd cDNA was 1495 bp long, encoding 395 amino acids. psdnd expression levels were investigated by real-time polymerase chain reaction (qRT-PCR) in various tissues and embryo developmental stages. psdnd transcripts were detected in the testes and ovaries, but not in somatic tissues. Embryonic psdnd expression levels were higher during early embryo development stages than during late embryogenesis; psdnd expression was highest at the 1 cell stage, then gradually decreased throughout the subsequent developmental stages. The spatial expression pattern was analyzed by whole-mount in situ hybridization (WISH). The psdnd transcripts migration pattern was similar with zebrafish (Danio rerio). Our results suggest that psdnd may function as a germ cell-specific marker.

Use of a gene encoding zona pellucida 4 as a female-specific marker for early stage sexual differentiation and size dimorphism in the pacific abalone Haliotis discus hannai.

Growth rates of Pacific abalone Haliotis discus hannai are an important trait affecting the economic value in the abalone aquaculture industry. A reverse-transcription polymerase chain reaction (RT-PCR) analyses of tissues from H. discus hannai was conducted for sexually mature gonads to determine male- and female-specific target gene expression, including genes encoding zona pellucida domain 4 (zp4), sperm protein (sp) and lysin (lys), respectively. Sex-specific expression patterns of these gene expression, even in sexually immature abalone, indicate these genes can be used as sensitive and robust sex-specific molecular markers. The RT-PCR procedure was also performed to analyze tissues collected at various developmental stages (50-day intervals) beginning at fertilization to determine when sex differentiation and expression of sex-specific genes was initiated. Detection of zp4 transcript in tissues collected at 200 days post-fertilization (dpf) indicated egg-specific development starts at 150-200 dpf. To evaluate possible sex-specific differences in growth rate, there was conducting of a molecular marker-based sex identification of abalone from a population selected for rapid growth rate. In a group of large H. discus hannai, females were more prevalent than males. To assess the correlation between growth and sex, there was comparison of weights of 3-year-old Pacific abalone in specimens where there had been sex determinations by visual examination and molecular methods. The results indicated females weighed more (55.92 ± 9.38 g, n = 15) than males (43.64 ± 15.55 g, n = 6, P = 0.037), indicating females had a more rapid growth rate than males.

Effects of Cryoprotective Medium Composition, Dilution Ratio, and Freezing Rates on Spotted Halibut (Verasper variegatus) Sperm Cryopreservation.

The spotted halibut is species that has a high potential market value in Korea, but the supply of seed is unstable because of the limited milt production of males. The objective of this research was to explore different aspects, such as CPAs, diluents, dilution ratio, and freezing rates, to develop an optimal sperm cryopreservation. The parameters assessed were movable sperm ratio, sperm activity index, survival rate, and DNA damage. The CPAs tested in this research were propylene glycol, dimethyl sulfoxide (DMSO), methanol, ethylene glycol, and glycerol. Different diluents, including 300 mM sucrose, 300 mM glucose, Stain's solution, and Ringer's solution, were investigated. The previous experiment showed that the optimal CPA for cryopreservation was DMSO with a concentration of 15% with 300 mM as diluent. To determine the effect of the dilution ratio, sperm was diluted to 1:1, 1:2, 1:10, 1:100, and 1:1000 with 300 mM sucrose containing DMSO at a final concentration of 15%. Lastly, the optimal freezing rate of the sperm was evaluated with four different freezing rates (-1, -5, -10, and -20 °C/min). Post-thaw sperm motility was higher with a dilution ratio lower than 1:2, and the freezing rate was less than -5 °C/min. In conclusion, these findings represent the development of a cryopreservation protocol for spotted halibut.

Cristazine, a novel dioxopiperazine alkaloid, induces apoptosis via the death receptor pathway in A431 cells.

The fungus Chaetomium sp. is a causative agent of infections in humans and is ubiquitous in the natural environment. The secondary metabolites of this genus exhibit many biological activities, including antifungal activity and toxicity in mitochondria. In this study, we isolated cristazine from the fungus C. cristatum, which has the potential to inhibit the growth of human epidermoid carcinoma (A431) cells in a dose- and time-dependent manner. Its inhibitory activity was examined using a cell viability assay and cell death was elucidated by western blot analysis. Cristazine triggered apoptotic cell death via the Type I death receptor pathway including the activation of caspases and other target proteins. However, cristazine did not have any effect on mitochondrial apoptotic proteins such as Bid, cytochrome c, and apoptosis-inducing factor. Cristazine inhibited the cell cycle progression by arresting the G1 /S phase and up-regulating the inhibitory proteins of cyclin-dependent kinases. Thus, cristazine has great potential for inducing apoptosis in A431 cells via both cell cycle arrest and the inhibition of cell growth.

Complete mitochondrial genomes of two Pleuronectid species: Clidoderma asperrimum and Verasper variegatus (Teleostei: Pleuronectiformes: Pleuronectidae).

Complete mitochondrial genomes of two Pleuronectid species, Clidoderma asperrimum and Verasper variegatus (Teleostei: Pleuronectiformes: Pleuronectidae) were analysed using the primer walking method. Their mitogenomes were 17,632 and 17,273 bp in total length, respectively and comprised 13 protein-coding genes, 2 ribosomal RNA genes, and 22 transfer RNA genes. Their gene contents and orders were similar to those of typical vertebrates. All Pleuronectid species were subdivided into three clades in the phylogenetic tree, and the two Pleuronectid species analysed in this study formed a strong monophyletic group comprising species belonging to three genera, Hippoglossus, Reinhardtius, and Verasper.

Multiple calmodulin genes of the Pacific abalone, Haliotis discus hannai (Mollusca: Vetigastropoda: Haliotidae).

In this study, we identified four canonical calmodulin genes in the Pacific abalone, Haliotis discus hannai. Their full-length cDNAs were variable in the 5' and 3' untranslated regions, but highly similar (91-97%) in the coding region. Each of the genes encoded 149 amino acids, with 93-97% similarity among themselves and 94-98% similarity with human CAM I. There were 54 substitutions distributed unevenly throughout the coding regions, found mostly in the third codon position. Gene structure analysis revealed that each of the calmodulin genes comprised five exons and four introns. The intron positions and phases were identical and there were no introns in the fourth exon. The corresponding introns differed in their sequences and sizes. Expression profiles of nine tissues from abalone revealed that the calmodulin genes were transcribed in common in gill and mantle tissue, but differentially in the other tissues. A phylogenetic analysis based on the amino acid sequences revealed that calmodulin C was the most common isoform in Gastropoda and calmodulin was the most diverged isoform. An in silico analysis of the calmodulin genes identified paralogous genes in other Haliotis species, indicating that gene duplication might have occurred in the last common ancestor of Haliotis. Abbreviations: ORF: open reading frame; RACE: random amplification of cDNA end; TSA: transcriptome shotgun assembly; UTR: untranslated region.

Effects of different diluents, cryoprotective agents, and freezing rates on sperm cryopreservation in Epinephelus akaara.

We investigated various factors, including cryoprotective agents (CPAs), diluents, and freezing rates, to develop an optimal cryopreservation protocol for Epinephelus akaara sperm. In experiments using 10% dimethyl sulfoxide (DMSO), glycerol, and methanol with various diluents, 10% DMSO and 300 mM glucose yielded the highest post-thaw sperm motility. The combination of 10% glycerol and 300 mM sucrose yielded significantly higher post-thaw sperm motility than did combinations using other diluents. Glycerol and DMSO at a concentration of 10% as CPAs with 300 mM glucose as the diluent resulted in the highest MSR and sperm activity index (SAI). An investigation to determine the effects of glycerol and DMSO concentrations on post-thaw sperm survival rate revealed no significant differences among 5, 10, 15, and 20% concentrations of either CPA. In assessing the effects of CPA concentration on the fertilization rate, the 10% concentration yielded the highest fertilization rate (81.4 ±â€¯4.3%) in DMSO, whereas 15% was the optimal concentration for glycerol (fertilization rate = 66.7 ±â€¯6.1%). The hatching rate was also highest in 10% DMSO (40.1 ±â€¯0.4%) and in 15% glycerol (27.8 ±â€¯2.3%). In conclusion, the optimal rates of post-thaw sperm motility, fertilization, and hatching were achieved when E. akaara sperm were cryopreserved in a diluent of 300 mM glucose with 10% DMSO as the CPA at a freezing rate of -5 °C/min. We therefore recommend this protocol for the cryopreservation of E. akaara sperm.

Molecular characterization of kappa class glutathione S-transferase from the disk abalone (Haliotis discus discus) and changes in expression following immune and stress challenges.

Glutathione S-transferase (GST; EC 2.5.1.18) isoenzymes represent a complex group of proteins that are involved in phase II detoxification in several organisms. In this study, GST kappa (GSTκ) from the disk abalone (Haliotis discus discus; AbGSTκ) was characterized at both the transcriptional and functional levels to determine its potential capacity to perform as a detoxification agent under conditions of different stress. The predicted AbGSTκ protein consists of 227 amino acids, with a predicted molecular weight of 25.6 kDa and a theoretical isoelectric point (pI) of 7.78. In silico analysis reveals that AbGSTκ is a disulfide bond formation protein A (DsbA), consisting of a thioredoxin domain, GSH binding sites (G-sites), and a catalytic residue. In contrast, no hydrophobic ligand binding site (H-site), or signal peptides, were detected. AbGSTκ showed the highest sequence identity with the orthologue from pufferfish (Takifugu obscurus) (60.0%). In a phylogenetic tree, AbGSTκ clustered closely together with other fish GSTκs, and was evolutionarily distanced from other cytosolic GSTs. The predicted three-dimensional structure clearly demonstrates that the dimer adopts a butterfly-like shape. A tissue distribution analysis revealed that GSTκ was highly expressed in the digestive tract, suggesting it has detoxification ability. Depending on the tissue and time, AbGSTκ showed different expression patterns, and levels of expression, following challenge of the abalone with immune stimulants. Enzyme kinetics of the purified recombinant proteins demonstrated its conjugating ability using 1-Chloro-2,4-dinitrobenzene (CDNB) and glutathione (GSH) as substrates, and suggested it has a low affinity for both substrates. The optimum temperature and pH for the rAbGSTκ GSH: CDNB conjugating activity were found to be 35 °C and pH 8, respectively indicating that the abalone is well adapted to a wide range of environmental conditions. Cibacron blue (100 µM) was capable of completely inhibiting rAbGSTκ (100%) with an IC50 (half maximal inhibitory concentration) of 0.05 µM. A disk diffusion assay revealed that rAbGSTκ could significantly protect cells from H2O2, CdCl2, and ZnCl2. Altogether, this current study suggests that AbGSTκ is involved in detoxification and immunological host defense mechanisms and allows abalones to overcome stresses in order for them to have an increased chance of survival.

Genome Sequences of a Novel Picorna-Like Virus from Pacific Abalone (Haliotis discus hannai) in South Korea.

We report the draft genome sequences of a novel member of the Picornavirales isolated from Pacific abalone (Haliotis discus hannai). The full length of the assembled draft genome sequences, obtained by use of a next-generation sequencing technique, were 8,019 nucleotides, including an RNA-dependent RNA polymerase gene (5,088 nucleotides) and a capsid protein gene (2,553 nucleotides). This genome sequence will be useful for understanding viral disease of Pacific abalone.

Characterization and spatiotemporal expression of gonadotropin-releasing hormone in the Pacific abalone, Haliotis discus hannai.

Gonadotropin-releasing hormone (GnRH) is a key neuropeptide regulating reproduction in humans and other vertebrates. Recently, GnRH-like cDNAs and peptides were reported in marine mollusks, implying that GnRH-mediated reproduction is an ancient neuroendocrine system that arose prior to the divergence of protostomes and deuterostomes. Here, we evaluated the reproductive control system mediated by GnRH in the Pacific abalone Haliotis discus hannai. We cloned a prepro-GnRH cDNA (Hdh-GnRH) from the pleural-pedal ganglion (PPG) in H. discus hannai, and analyzed its spatiotemporal gene expression pattern. The open reading frame of Hdh-GnRH encodes a protein of 101 amino acids, consisting of a signal peptide, a GnRH dodecapeptide, a cleavage site, and a GnRH-associated peptide. This structure and sequence are highly similar to GnRH-like peptides reported for mollusks and other invertebrates. Quantitative polymerase chain reaction demonstrated that Hdh-GnRH mRNA was more strongly expressed in the ganglions (PPG and cerebral ganglion [CG]) than in other tissues (gonads, gills, intestine, hemocytes, muscle, and mantle) in both sexes. In females, the expression levels of Hdh-GnRH mRNA in the PPG and branchial ganglion (BG) were significantly higher at the ripe and partial spent stages than at the early and late active stages. In males, Hdh-GnRH mRNA levels in the BG showed a significant increase in the partial spent stage. Unexpectedly, Hdh-GnRH levels in the CG were not significantly different among the examined stages in both sexes. These results suggest that Hdh-GnRH mRNA expression profiles in the BG and possibly the PPG are tightly correlated with abalone reproductive activities.

Differentially-Expressed Genes Associated with Faster Growth of the Pacific Abalone, Haliotis discus hannai.

The Pacific abalone Haliotis discus hannai is used for commercial aquaculture in Korea. We examined the transcriptome of Pacific abalone Haliotis discus hannai siblings using NGS technology to identify genes associated with high growth rates. Pacific abalones grown for 200 days post-fertilization were divided into small-, medium-, and large-size groups with mean weights of 0.26 ± 0.09 g, 1.43 ± 0.405 g, and 5.24 ± 1.09 g, respectively. RNA isolated from the soft tissues of each group was subjected to RNA sequencing. Approximately 1%-3% of the transcripts were differentially expressed in abalones, depending on the growth rate. RT-PCR was carried out on thirty four genes selected to confirm the relative differences in expression detected by RNA sequencing. Six differentially-expressed genes were identified as associated with faster growth of the Pacific abalone. These include five up-regulated genes (including one specific to females) encoding transcripts homologous to incilarin A, perlucin, transforming growth factor-beta-induced protein immunoglobulin-heavy chain 3 (ig-h3), vitelline envelope zona pellucida domain 4, and defensin, and one down-regulated gene encoding tomoregulin in large abalones. Most of the transcripts were expressed predominantly in the hepatopancreas. The genes identified in this study will lead to development of markers for identification of high-growth-rate abalones and female abalones.

Molecular Cloning and Co-Expression of Phytoene Synthase Gene from Kocuria gwangalliensis in Escherichia coli.

A phytoene synthase gene, crtB, was isolated from Kocuria gwangalliensis. The crtB with 1,092 bp full-length has a coding sequence of 948 bp and encodes a 316-amino-acids protein. The deduced amino acid sequence showed a 70.9% identity with a putative phytoene synthase from K. rhizophila. An expression plasmid, pCcrtB, containing the crtB gene was constructed, and E. coli cells containing this plasmid produced the recombinant protein of approximately 34 kDa , corresponding to the molecular mass of phytoene synthase. Biosynthesis of lycopene was confirmed when the plasmid pCcrtB was co-transformed into E. coli containing pRScrtEI carrying the crtE and crtI genes encoding lycopene biosynthetic pathway enzymes. The results obtained from this study will provide a base of knowledge about the phytoene synthase of K. gwangalliensis and can be applied to the production of carotenoids in a non-carotenoidproducing host.

Evaluation of extenders and cryoprotectants on motility and morphology of longtooth grouper (Epinephelus bruneus) sperm.

The objective was to evaluate various extenders and cryoprotectants on postthaw motility of longtooth grouper (Epinephelus bruneus) sperm, based on sperm motility ratio (SMR), sperm velocity (SV), and morphological damage after thawing. To evaluate the optimal cryoprotectant for cryopreservation of longtooth grouper sperm, semen was diluted in 0.3 M glucose extender containing one of four cryoprotectants (dimethyl sulfoxide, methanol, ethylene glycol, and glycerol [Gly]) at a final concentration of 10% or 20%, and then frozen in liquid nitrogen vapor (-76 °C for 3 minutes) before storage in liquid nitrogen. Semen diluted in 0.3 M glucose containing 10% Gly had the highest postthaw SMR (57.5 ± 2.5%, mean ± SEM). To identify the optimal extender, semen was diluted in one of two longtooth grouper artificial seminal plasma (LG-ASP; LG-ASP1 and LG-ASP2) extenders containing Gly at concentrations of 0%, 5%, 10%, or 20%, then cryopreserved using the described procedure. Semen diluted with LG-ASP2 extender containing 10% Gly cryoprotectant had the best postthaw SMR (66.3 ± 2.0%) and SV (135.9 ± 4.5 µm/s). Compared with fresh sperm, some structural damage was observed in cryopreserved sperm. We concluded that the combination of LG-ASP2 and 10% Gly (as extender and cryoprotectant, respectively), resulted in high postthaw SMR and SV for cryopreservation of longtooth grouper sperm.

Characterization of genes encoding prolactin and prolactin receptors in starry flounder Platichthys stellatus and their expression upon acclimation to freshwater.

This study aims to investigate the genes encoding prolactin (PRL) and prolactin receptors (PRLR) and their tissue-specific expression in starry flounder Platichthys stellatus. Starry flounder PRL gene consisting of five exons encodes an ORF of 212 amino acid residue comprised of a putative signal peptide of 24 amino acids and a mature protein of 188 amino acids. It showed amino acid identities of 73 % with tuna Thunnus thynnus, 71 % with black porgy Acanthopagrus schlegelii, 69 % with Nile tilapia Oreochromis niloticus, 64 % with pufferfish Takifugu rubripes, 63 % with rainbow trout Oncorhynchus mykiss, and 60 % with mangrove rivulus Kryptolebias marmoratus. Phylogenetic analysis of piscine PRLs also demonstrated a similarity between starry flounder and other teleosts but with a broad distinction from non-teleost PRLs. PRLR gene consists of eight exons encoding a protein of 528 amino acid residues. It showed a similarity to the PRLR2 subtype as reflected by amino acid identities of 54 % with A. schlegelii, 48.1 % with K. marmoratus, 46.3 % with tilapia O. mossambicus, and 46.1 % with O. niloticus PRLR2 as compared to PRLR1 isoform having less than 30 % identities. While mRNA transcript corresponding to PRL was detected only from the pituitary, most of PRLR mRNA was detected in the gill, kidney, and intestine, with a small amount in the ovary. The level of PRL transcript progressively increased during 6 days of acclimation to freshwater and then decreased but stayed higher than that of seawater at 60 days of acclimation. An opposite pattern of changes including a decrease at the beginning of the acclimation but a slight increase in the level osmolality was found as adaptation continued. The results support the osmoregulatory role of PRL signaling in starry flounder.