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The residual estrogenic pollutant alkyl phenols, 4-nonylphenol (NP), 4-tert-octylphenol (OP), and the sex ratio of Formosa landlocked salmon Oncorhynchus formosanus were surveyed in the Formosa landlocked salmon ecological reservation area. This species survives as a remnant, landlocked population in the headwaters of the Chichiawan Stream, a tributary of the Tachia River of central Taiwan. Dams restrict the movement of fish in and out of the agriculturally influenced zones. Fish (n = 157) were collected from a total of 9 habitat sites, and the amount of NP and OP present in each site (ambient water from rearing pools, streams, stream sediments, and agricultural effluents) was measured and the sex of the fish was determined via a noninvasive PCR-based method. The results indicate significantly higher NP content and a skewed sex ratio in Formosa landlocked salmon found in agricultural zones compared to salmon found in non-agricultural zones. The concentrations and distributions of NP observed in this study are ecologically significant because they correspond with the skewed sexual ratio and the lower quality and quantity of male salmon sperm observed in previous field investigations. Although the skewed sex ratios are likely a reflection of multiple endocrine disrupting compounds rather than simply NP and OP alone, these results support the contention that exposure of Formosa landlocked salmon to environmentally persistent estrogenic chemicals can have deleterious reproductive consequences.

Oncorhynchus masou formosanus (Formosa landlocked salmon) is a critically endangered salmonid fish endemic to Taiwan. To begin to understand how its drastic change in lifestyle from anadromous to exclusively river-dwelling is reflected in its immune genes, we characterized the genes encoding six cytokines (IL-2A, IL-2B, IL-4/13A, IL-4/13B1, IL-4/13B2, and IL-17A/F2a) important for T cell responses as no genomic data is available for this fish. Interestingly, all genes appeared homozygous indicative of a genetic bottleneck. The IL2 and IL17A/F2a genes and their products are highly similar to their characterized homologs in Oncorhynchus mykiss (rainbow trout) and other salmonid fish. Two notable differences were observed in IL4/13 family important for type 2 immune responses. First, O. m. formosanus carries not only one but two genes encoding IL-4/13B1 proteins and expansions of these genes are present in other salmonid fish. Second, the OmfoIL4/13A gene carries a 228 bp deletion that results in a premature stop codon and hence a non-functional IL-4/13A cytokine. This suggests a reduced ability for T cell responses against parasitic infections in this species., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

We investigated the effects of cryopreservation on the quality of Portuguese oyster (Crassostrea angulata) sperm, which were examined before and after freezing; sperm motility, fertilizing capacity, and ultrastructural morphology were analyzed. The motility percentage and fertilizing capacity of the cryopreserved sperm (mean ± standard error) were 16% ± 1% and 17% ± 8%, respectively. In the pre-freezing sperm, these were 58% ± 2% and 76% ± 4%, respectively. The sperm sustained substantial morphological and ultrastructural damage during cryopreservation. The morphological changes varied considerably in nature and extent, ranging from no apparent damage to virtual disintegration. Sperm were stained with fluorescent dyes to assess viability, plasma membrane integrity, mitochondrial activity, acrosomal membrane integrity, oxidation level, and DNA fragmentation and examined through flow cytometry. The methods used for the flow cytometry assays were slightly modified from those used for evaluating the semen quality of livestock. Relative to the pre-freezing sperm, the frozen-thawed sperm exhibited lower acrosomal membrane integrity (acrosomal damage, 59.86 ± 5.29; P < 0.05) and substantially higher oxidation levels (free radicals, 60.06 ± 0.82; P < 0.003). Oxidation level was found to be the most sensitive indicator of cryodamage. Along with ultrastructural analysis, we used flow cytometry to measure the qualitative and quantitative characteristics of Portuguese oyster sperm before and after cryopreservation rapidly, objectively, and accurately. This is the first study to assess the quality of Portuguese oyster sperm through these methods., (Copyright © 2021 Elsevier Inc. All rights reserved.)

In the present study, we have investigated the ultrastructures of the mature gonadal spermatozoa of R. variegata and T. literatus and presented comparisons with the Manila clam, R. philippinarum, sperm ultrastructure examined. Spermatozoa of R. variegata consist of (in anterior to posterior sequence): an elongate conical, deeply invaginated, acrosomal vesicle (length 1.58 ± 0.06 μm; width 0.99 ± 0.07 μm; invagination occupied by a granular subacrosomal material); a barrel-shaped nucleus (length 1.82 ± 0.06 μm; width 1.50 ± 0.03 μm); a midpiece consisting of two orthogonally arranged centrioles, surrounded by four spherical mitochondria; nine satellite fibers connecting the distal centriole to the plasma membrane; and a flagellum originating from the distal centriole. Contents of the acrosomal vesicle of R. variegata are differentiated into a very electron-dense basal ring and a less electron-dense zone (with seven dense transverse layers structure) on the anterior region of the acrosome. Spermatozoa of T. literatus differ from those of R. variegata and are characterized by a rounded-conical invaginated, acrosomal vesicle (length 0.88 ± 0.08 μm; width 0.77 ± 0.06 μm), with a basal ring; and an anteriorly-tapered, barrel-shaped nucleus (length 1.57 ± 0.04 μm; width 1.60 ± 0.09 μm); a midpiece composed of four mitochondria. Centriolar and flagellar details are essential as for R. variegata. Sperm morphology separating R. variegate, R. philippinarum, and T. literatus in different clades. The anterior region of the acrosomal vesicle in R. variegata sperm had the transverse bands structure whereas the apex of the acrosomal vesicle of T. literatus sperm had no such structure. This difference advocated that acrosomal feature could be an important character for taxonomic distinction. Our data supported the previous studies that the ultrastructure of bivalve sperm is species-specific. This advocates that the phyletic relationships of Tapetinae, commonly based on shell morphology, should also add additional and newer approaches., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Spermatozoan ultrastructure and complete mitochondrial genome in the marine bivalve mollusk Meretrix sp. (Taiwan) from Taiwan are described and contrasted with other bivalves, especially within Meretrix. We have examined the features of the mature gonadal spermatozoa of Meretrix sp. (Taiwan) and provided comparisons with the other four Meretrix species (M. petechialis, M. meretrix, M. lyrata, and M. lamarckii). The morphological characteristics of these spermatozoa are diagnostic for each of the species studied here. The most marked interspecific difference was found in the acrosome. Meretrix sp. (Taiwan) is genetically distinct and is a different species from M. petechialis and M. lusoria (Japan) based on complete mitochondrial genome data. Sperm data for Meretrix are limited but show remarkable congruence with the molecular results. We suggest use Meretrix formosa Gwo and Hsu as the scientific name for Taiwanese hard clams, Meretrix sp. (Taiwan). Additional species, particularly the Japanese hard clam (M. lusoria) require examination before this tentative conclusion can be verified., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Small abalone (Haliotis diversicolor) is a commercially valuable species for both fisheries and aquaculture. The production of annual farmed small abalone in Taiwan, once the highest in the world, has dramatically decreased in the past 15 years, and currently, the industry is close to collapse. Understanding the genetic diversity of small abalone and developing stock identification methods will be useful for genetic breeding, restoring collapsed stocks, managing stocks, and preventing illegal trade. We investigated 307 cultured and wild individuals from Taiwan, Japan, and Bali Island (Indonesia) by using the mitochondrial cytochrome c oxidase subunit I (COI) gene. Network analysis of mtDNA COI gene sequences revealed that the individuals collected from Taiwan, Japan, and Indonesia could be identified, and showed significant genetic divergence. In addition, the Indonesian population (Haliotis diversicolor squamata) was significantly different from the other populations and might need to be considered a separate species. We discovered a single nucleotide polymorphism marker in the mtDNA COI gene that can be used to distinguish the Taiwan population from the Japan population. We also developed a polymerase chain reaction-restriction fragment length polymorphism method for rapid detection. Furthermore, we could identify the cultured stocks, wild population, and hybrid stocks by using 6 microsatellites and amplified fragment length polymorphism. This study contributes useful tools for stock identification and the production of high-disease resistant small abalone strains (Japan × Taiwan or Taiwan × Japan). Efforts should be made to avoid unintentional random genetic mixing of the Taiwan population with the Japan population and subsequent breakdown of population differentiation, which impair local adaptation of the Taiwan wild population. Molecular markers revealed a split between the Taiwan and Japan populations, and the existence of a possible barrier to the free dispersal of small abalone is discussed.

This study sampled six times of river water, sediment, and tilapia (Oreochromis niloticus) in the Dan-Shui River, Taipei, Taiwan; 10 feminizing compounds were analyzed using ultra-performance liquid chromatography-tandem mass spectrometry. Bisphenol A (508 ± 634 ng/L, geometric mean (GM) 303 ng/L) and nonylphenol (491 ± 570 ng/L, GM 328 ng/L) were the most abundant among analytes in the river water. Nonylphenol (770 ± 602 ng/g wet weight, GM 617 ng/g wet weight) was also the highest in sediment. Fish may uptake nonylphenol and nonylphenol ethoxylates from river water and sediment because there were significant correlations between the concentrations in these matrixes and those in fish tissues (r s ranged from 0.21 to 0.49, p < 0.05). The bioaccumulation of nonylphenol, nonylphenol ethoxylates and bisphenol A in gonad, eggs, and liver was much higher than that in muscle (e.g. mean bioaccumulation factors of nonylphenol were 27,287, 20,971, 9,576 and 967, respectively) and might result in low liver fractions in fish body weights (0.66 % ± 0.39 %, GM 0.55 %) and the skewed sex ratio of fish (male to female = 0.52). This innovative study linked the environmental and internal doses statistically in the globally distributed wild fish by analyzing feminizing compounds in water, sediment, and four fish tissues including gonad and eggs.

Peanut worm (Sipunculus nudus) is a cosmopolitan species mainly distributed in tropical and subtropical coastal waters. Analysis of the mitochondrial cytochrome c oxidase subunit I (COI) gene sequences among S. nudus from GenBank revealed high genetic variation (p-distance, 0.115-0.235; k2p, 0.128-0.297) and paraphyletic relationships. These indicated misidentification and/or cryptic diversity may be present in the genus Sipunculus. To understand the genetic diversity and to manage the recourse of S. nudus, we collected specimens from coastal waters of southern China and Taiwan. In the phylogenetic topology, specimens can be separated into four distinct clades; three of these clades (clade A, B and C) were only represented from this region (southern China and Taiwan), but the clade D grouped with individuals from Central America (Atlantic coast). Furthermore, individuals of clades A and D were collected at the same location, which does not support the hypothesis that this genetic break reflects contemporary geographical isolation. The four distinct clades observed among coastal waters of southern China and Taiwan indicated underestimated diversity. It is noteworthy that the cryptic diversity is vulnerable under high pressure of human activity., (© 2013 John Wiley & Sons Ltd.)

Papaya (Carica papaya L.) is established as a cash crop throughout the tropical and subtropical regions due to its easy adaptation to diverse agricultural conditions, high yields, and prompt returns. The sex types of papaya plants are hermaphrodite, male, and female. Among them, hermaphroditic plants are the major type in papaya production, because the fruit has commercial advantages over that of the other sexes. Sex inheritance in papaya is determined by the M and M(h) dominant alleles in males and hermaphrodites, respectively, and a recessive m allele in females. Currently, all hermaphrodite seeds are not available due to the lethality of dominant homozygosity. Therefore, in this study, six male-hermaphrodite-specific markers were developed for a rapid sex identification using multiplex loop-mediated isothermal amplification (mLAMP) to efficiently and precisely select hermaphroditic individuals in the seedling or early growth stage. The LM1-LAMP assay consisted of two sex-LAMP reactions for amplifying two male-specific markers (T12 and Cpsm90) in one reaction, and showed several advantages in terms of a rapid reaction time (<1 h), isothermal conditions (less equipment required), a high efficiency (0.5 ng of DNA required in the reaction mixture), and an economical reaction system (5 μl in volume). The established method can be easily performed in the field by visual inspection and facilitates the selection of all hermaphroditic individuals in papaya production.

In mammals, androgens appear to enhance the development of primary ovarian follicles, but PI3K (phosphoinositide 3-kinases) pathway is well recognized as one of the critical pathways in early follicular development. Roles of the PI3K were revealed by deletion of PTEN (phosphatase and tensin homolog on chromosome 10). PTEN is demonstrated to play an important role in the early stage of follicle development. In the Japanese eel, two forms of PTEN have been cloned, but what their functions on the development of early ovarian follicles are still not clear. The natural blockage and inducible of ovarian development was a benefit to address this question in the eel. Testosterone (T) shows to ameliorate the early ovarian development in the eel. The aims of this study were to elucidate the two forms of PTEN by cellular and physiological criteria and to study the effects of T on the ovarian PTEN production in the exogenous pituitary extracts-stimulated eel. Our results suggested that two forms of PTEN are existing in the Japanese eel, and eel ovarian development corresponded to the decrease in ovarian PTEN expression, vice versa. In addition, the supplement of T on eel early ovarian development can be attributed to its PTEN inhibitor role.

This study developed and validated a method of measuring the feminizing chemicals 4-tert-octylphenol, 4-nonylphenol, nonylphenol monoethoxycarboxylate (NP(1)EC), nonylphenol monoethoxylate (NP(1)EO), nonylphenol diethoxylate (NP(2)EO), estrone, 17β-estradiol, estriol, 17α-ethinyl estradiol and bisphenol A in river water, sediment, and tissue using ultra-high performance liquid chromatography/tandem mass spectrometry (UHPLC/MS/MS) and isotope-dilution techniques. Water samples were pretreated using disk-type automated solid-phase extraction (SPE). Solid samples of sediment, fish, and clams were treated with matrix solid-phase dispersion (MSPD) using C(8) adsorbent. Eluents were directly passed following alumina cartridges for cleanup. The signal intensity of analytes on electrospray ionization (ESI) was compared with that of atmospheric pressure photoionization (APPI). The analytes were separated on a UHPLC C(18) column with aqueous 10-mM ammonium acetate for NPEOs and aqueous 10-mM N-methylmorpholine for the other compounds. On-line cleanup was evaluated using two-dimensional liquid chromatography (2-D LC). ESI could provide satisfactory response for all of the analytes. Though APPI did not offer suitable response for NP(1)EO, NP(2)EO and NP(1)EC, it provided better signal intensities for the steroid estrogens (1.0-2.4 times) and the phenols (3.2-4.4 times) than ESI. UHPLC shortened chromatographic time to less than 10 min. Disk-type automated SPE and MSPD dramatically increased the throughput of sample preparation. The extraction efficiency on surface water samples ranged from 10% to 91%. The extraction efficiency of MSPD on sediment, fish, and clams was 51-101%, 36-109%, and 30-111%, respectively. Acidic alumina cleanup was essential for the analysis of the tissue sample, and reduced matrix effects better than 2-D LC on-line cleanup. The limits of detection (LODs) in water ranged from 0.81 ng/L to 89.9 ng/L. The LODs in sediment and tissue ranged from tens of pg/g wet weight to only a few ng/g wet weight. This method proved to be accurate and reproducible, as both quantitative biases and relative deviations remained smaller than 20% at three spiked levels., (Copyright © 2011 Elsevier B.V. All rights reserved.)

In this study, DNA analyses were employed to verify the identity of six morphologically similar species that occur in the coastal waters of Taiwan: the black seabream complex (Acanthopagrus latus, Acanthopagrus schlegelii, Acanthopagrus sivicolus, Acanthopagrus taiwanensis, Acanthopagrus chinshira and Acanthopagrus pacificus). Amplified fragment-length polymorphism (AFLP) analyses clearly distinguished the same six species that are morphologically diagnosable based on subtle differences in scale counts and anal-fin colouration. In contrast, mitochondrial DNA analyses based on cytochrome b gene sequences did not distinguish individuals of A. schlegelii and A. sivicolus, reflecting either historical introgression or recent speciation and incomplete sorting of their mitochondrial lineages. Phylogenetic relationships among these six north-west Pacific Ocean species of Acanthopagrus analysed using AFLP data were consistent with scale rows above the lateral line (TRac), sperm ultrastructure and geographical distribution. The study provides molecular tools for future research relevant to improved management of these resources, and an increased understanding of the evolutionary history of this radiation., (© 2011 The Authors. Journal of Fish Biology © 2011 The Fisheries Society of the British Isles.)

Mottled spinefoot Siganus fuscescens and white-spotted spinefoot Siganus canaliculatus are two similar species that differ subtly in colouration and morphology. Three major mtDNA clades were identified for these species, but individuals were clustered by amplified fragment length polymorphism (AFLP) according to geography rather than morphology, suggesting that the colour morphs are interbreeding., (© 2011 The Authors. Journal of Fish Biology © 2011 The Fisheries Society of the British Isles.)

Species and sex identification are among the most important parameters for conservation management. However, it is extremely difficult to perform such identification in Formosa landlocked salmon (Oncorhynchus masou formosanus). Both sexual dimorphism in landlocked dwarf form Formosa landlocked salmon and morphological difference among cherry salmon complex are minimal. We developed a simple, rapid and noninvasive method for identifying sex and species of this critically endangered species using a loop-mediated isothermal amplification (LAMP) technique. The LAMP assay showed the advantage of simple detection (evaluated by visual inspection), rapid reaction time (< 1 h), isothermal condition (less equipment required) and high efficiency (only 0.5-5 pg of DNA was required in the reaction mixture). Therefore, the method is more economical and practical than PCR. The LAMP assay can be easily performed in the field and is a valuable tool for detecting sex ratios in wild populations and identifying species in commercial imports. This is the first application of LAMP in identifying species and sex of salmonids as far as we know and clearly shows the potential application of LAMP in molecular ecology and conservation efforts., (© 2011 Blackwell Publishing Ltd.)

Traditional mitochondrial 16S rRNA is commonly used in many species identification studies. However, it is difficult to apply to the phylogenetic studies among the Oncorhynchus subspecies, which is a crucial need for management purposes for Oncorhynchus masou formosanus, Taiwan salmon. In this study, we have developed an improved species identification method for Taiwan salmon distinguished with other Oncorhynchus subspecies tested by exploiting PCR for growth hormone (GH) 1 gene. By comparing DNA sequences for GH1 from 11 species of Oncorhynchus subspecies we designed novel PCR primers that exploit differences between Taiwan salmon and other Oncorhynchus subspecies. Therefore, the technique is an important tool in the management of populations of the endangered land-locked Taiwan salmon preventing from their possible hybrids with other Oncorhynchus subspecies once tested.

The general sperm structure of the investigated clupeoids possess an oliviform head with a distinct deep nuclear fossa, a midpiece with one mitochondrion and a posterior flagellum. They are characterized by two apomorphies: an annular or C-shaped mitochondrion and ITDs (intratubular differentiation) in the A tubules of the axonemal doublets. The fine structure of the spermatozoa of blue sprat, Spratelloides gracilis, was investigated to see if resulting data conformed to the current hypotheses of the taxonomy of the Clupeidae. The mature sperm is characterized by the following features: (1) the nucleus is oliviform; its prominent deep nuclear fossa encloses the initial portion of flagellum, (2) a proximal centriole has not been identified, (3) a single spherical mitochondrion is located laterally in relation to the flagellum and (4) no cytoplasmic canal is present. Our blue sprat-spermatozoan morphology data suggest that the blue sprat has Clupeomorpha affinities, while indicating no close affinity with Elopomorpha. Clupeoid sperm exhibit morphological variations with the nucleus and mitochondrion being particularly variable. This study provides useful systematic characters to the existing knowledge of comparative spermatology and may provide additional clue to Euteleost phylogeny.

The cryopreservation of algae could prevent genetic drift and minimize labor costs compared to the current method of maintenance and subculturing. Clear, simple protocols for cryopreservation of marine microalga, Nannochloropsis oculata were developed and cryoprotectant choice and concentration optimized. The viability of the microalga was assessed directly after thawing, and algal concentration was measured after 2-30 days of growth. Five cryoprotectants (dimethyl sulphoxide, Me2SO; ethylene glycol, EG; glycerol, Gly; methanol, MeOH; and propylene glycol, PG) at five concentrations (10, 20, 30, 40, and 50%; v/v) were evaluated to determine the toxicity of various cryoprotectants to N. oculata. The toxicity of cryoprotectant (Me2SO, EG, MeOH, and PG) was observed only at higher concentrations of CPAs: > 20% for EG, > 30% for Me2SO and methanol, and > 40% for PG. Direct freezing of algae in liquid nitrogen resulted in a severe loss of viability and a modified cryopreservation protocol proved to be more appropriate for the preservation of N. oculata. Cryopreservation protocols developed and tested in the present study might be applied to cryopreserving other strains, or species, in this genus.

The mature spermatozoa of two Taiwan protandrous hermaphrodite Sparidae Acanthopagrus berda and Acanthopagrus australis are investigated and compared with those of other two Sparidae (Lagodon rhomboids and Archosargus probatocephus) from the Western hemisphere. Ultrastructurally the spermatozoon of these four species has a spherical, homogeneously electron-dense nucleus with an axial nuclear fossa. The midpiece contains one to four spherical mitochondria and encircles the basal body of the flagellum. The mature spermatozoa of the four species are of the primitive or ect-aquasperm form and conform to the teleostean type I spermatozoon with the flagellar axis inserts perpendicular and medial to the nuclear fossa. Variation in the depths of the nuclear fossa and mitochondria number is substantial in these four Sparidae species. This study provide useful systematic characters to the existing knowledge of comparative spermatology of Sparidae.

Transmission and scanning electron microscopy were used to investigate the ultrastructure of spermatozoa in two Sparinae species Pagrus major and Rhabdosargus sarba. Ultrastructurally, the spermatozoa of P. major and R. sarba both consist of a spherical, homogeneously electron-dense nucleus with a deep axial nuclear fossa, and an unusual notch, in the nuclear region. The midpiece contains two spherical mitochondria in R. sarba and one in P. major. The comparison of spermatozoal ultrastructure of these two species of Sparidae shows that they closely resemble one another and suggests that they are closely related. Variation in the geometry and dimensions of the mitochondrion and nucleus is substantial in these two Sparidae species. It is concluded that the spermatozoa of both species are of primitive type, and they are distinguished by several unique features which may provide useful systematic characteristics.

Mature spermatozoa of two perciform teleost fishes, Paraupeneus spilurus (Mullidae) and Siganus fuscescens (Siganidae) from Taiwan were examined using transmission and scanning electron microscopy. Despite the fact that spermatozoa of both species are of the primitive type, the results of the present study highlight the potential application of spermatozoal morphology in studies of fish phylogenetic relationships. To our knowledge, the flattened nucleus observed in P. spilurus spermatozoa is reported for the first time. Several features common to Sigandae spermatozoa-the unusual almost parallel situation of the centrioles, the arrangement of mitochondria and the near absence of shallow nuclear fossa-are significantly different from other common teleost sperm types. These unique features may be synapomorphies for the Siganidae and Mullidae and evidently contribute to the study of phylogenetic relationships in teleosts.

We examined the applicability of the comet assay (single cell gel electrophoresis assay) to estimate the quality of frozen-thawed Pacific oyster (Crassostrea gigas) spermatozoa. Comet assay was performed on semen before and after cryopreservation followed by fluorescent staining with propidium iodide to assess DNA integrity. After cryopreservation, the percentage of spermatozoa with damaged DNA significantly increased, while only about half of the cells displayed intact DNA, even when protected with 10 percent DMSO. All the considered parameters (head length, head area, head intensity, total length, total area, total intensity, tail length percent, tail area percent, and tail intensity percent) were higher than the oyster sperm protected with 10 percent DMSO-artificial sea water after freezing and thawing. Only tail length percent, tail area percent, and tail intensity percent were increased significantly after cryopreservation. The tail length percent was found to be the most sensitive indicator of the cryopreservation-induced DNA damage. Our freeze-thawing procedure significantly affected oyster sperm DNA, as indicated by the reduced fertilization rate when frozen-thawed oyster sperm are used. Irreversible alteration of the genome may prevent fertilization or alter normal embryonic development. This study is the first to demonstrate that the comet assay is an inexpensive, rapid and sensitive method for determining DNA damage in Pacific oyster sperm quality assessments.

Methods for cryopreserving spermatozoa and maximizing fertilization rate in Taiwan small abalone, Haliotis diversicolor supertexa, were developed. The gametes (spermatozoa and eggs) of small abalone were viable 3 h post-spawning, with fertilization, and development rate decreasing with time. A minimum of 10(2) cell/ml sperm concentration and a contact time of 2 min between gametes is recommended for artificial insemination of small abalone eggs. Eight cryoprotectants, dimethyl sulfoxide (DMSO), dimethyl acetamide (DMA), ethylene glycol (EG), propylene glycol (PG), butylene glycol (BG), polyethylene glycol, glycerol and methanol, were tested at concentrations between 5 and 25% to evaluate their effect on motility of spermatozoa exposed to cryoprotectant for up to 60 min at 25 degrees C before freezing. The least toxic cryoprotectant, 10% DMSO, was added to artificial seawater (ASW) to formulate the extender for freezing. Semen was diluted 1:1 with the extender, inserted into 1.5 ml microtubes and frozen using a cooling rate between -3.5 and -20 degrees C/min to various transition temperatures (0, -30, -60, -90 and -120 degrees C), followed by transfer and storage in liquid nitrogen (-196 degrees C). The microtubes were thawed from +45 to +145 degrees C/min. Spermatozoa, cooled to -90 degrees C at a cooling rate of -12 or -15 degrees C/min and then immersed in liquid nitrogen, had the best post-thaw motility. Post-thaw sperm motility was markedly reduced compared to fresh sperm. More frozen-thawed spermatozoa are required to achieve fertilization rates comparable to those achieved using fresh spermatozoa.

Using transmission and scanning electron microscopy, the mature spermatozoa of four bivalves of the family Veneridae--Gafrarium tumidum and Circe scripta (Circinae), Pitar sulfureum (Pitarinae) and Gomphina aequilatera (Tapetinae)--are described for the first time and compared with those of other bivalves, particularly other heterodonts. As our observations show, the spermatozoa of these four species are of the primitive type or ect-aquasperm form. The head contains a slightly curved nucleus with a short cone-shaped acrosome. The structure of the acrosome is typical of heterodont bivalves and two major components of the acrosomal vesicle material can be distinguished. The midpiece exhibits four or five mitochondria which surround the proximal and the distal centrioles. Variation in the shape and dimensions of the acrosomal vesicle and nucleus is substantial in these four Veneroidea species.

Using transmission and scanning electron microscopy, we have described the mature spermatozoon of the bivalve Gafrarium tumidum (Heterodonta, Veneridae, Circinae) for the first time. The spermatozoon of G. tumidum is the ect-aquasperm type. The head contains a slightly curved nucleus. At its apex is a short cone-shaped acrosome. The structure of the acrosome is typical of heterodont bivalves and two major components of the acrosome vesicle material can be distinguished. The midpiece is an annular band of five mitochondria which surround the centriole complex. Sperm ultrastructure of G. tumidum provides additional information for the existing Veneridae phylogeny.

The Formosan landlocked salmon (Oncorhynchus masou formosanus) are at a high risk of extinction, and the sustained maintenance of the population will soon depend on aquaculture systems, which use cryopreservation of spermatozoa to increase genetic diversity. We investigated the effectiveness of dimethyl sulfoxide (DMSO), dimethyl-acetamide (DMA), and methanol as cryoprotectants in combination with 300 mM glucose as extender on the freezing of Formosan landlocked salmon spermatozoa. We also evaluated the morphological changes of Formosan landlocked salmon spermatozoa after their immediate dilution in the 300 mM glucose-DMSO extender and after freeze-thawing. The spermatozoa frozen with DMSO as a cryoprotectant showed significantly higher post-thaw motility and fertility than spermatozoa frozen with DMA or methanol. The fertilization capacity of frozen-thawed Formosan landlocked salmon was comparable to that of fresh spermatozoa. Intersubspecies fertilization trials between cryopreserved Formosan landlocked salmon spermatozoa and Amago salmon eggs showed high fertilization rates. Based on the findings, the potential value of using sperm bank to safeguard this endangered species is discussed.

To improve availability of penaeid seedstock during periods of high demand, experiments were conducted to determine the feasibility of stockpiling embryos by freezing them. Embryos were screened for developmental stage; cryoprotectants, chilling effects, and freezing regimens were likewise evaluated. Juvenile forms (embryos, nauplii and zoea) of Penaeus japonicus were exposed to various cryoprotectants, including dimethyl sulfoxide, glycerol, methanol, ethylene glycerol and polyethylene glycol 300 under ambient temperature (25 degrees C). Following this bioassay, maximum safe concentrations of each cryoprotectant were tested on the juveniles under chilling to 0 degree C and with 42 freezing regimens. Methanol was found to be relatively nontoxic. Early developmental stages were the most sensitive to chilling. Initial attempts to freeze P. japonicus juveniles were reported. The survival rate of nauplii and zoea treated with 10% methanol in natural sea water (35 ppt salinity) and frozen to -15 degrees C was 85%, and some nauplii and zoea survived freezing to -25 and -196 degrees C. However, no treatment yielded normal nauplii or zoea after freezing.

With particular emphasis on mitochondria that may provide endogenous energy for spermatozoan motility, the morphological changes of the spermatozoa of three marine teleosts, black porgy (Acanthopagrus schlegelli), black grouper (Epinephelus malabaricus), and Atlantic croaker (Micropogonias undulatus), were compared either after activation in artificial sea water or when immersed in various osmotic pressure media. The midpieces of these three teleosts spermatozoa are composed of mitochondria surrounding the flagellum. Each mitochondrion is enclosed by distinct outer and inner membranes. The inner membrane separates the organelle's volume into two phases: the matrix and the intermembrane space. The inner membrane displays numerous infolding cristae that vary in number and shape and extend into the matrix. Following activation with artificial sea water, spermatozoa became motile and both the size and number of mitochondria decrease and then totally disappear. The present study strongly suggests that an energy source(s), responsible for motility, is located within the mitochondria in the midpiece of these three marine teleost spermatozoa.

The effects of mechanical shock and thermal stress on red drum (Sciaenopus ocellatus) embryos at specific developmental stages were studied. The tolerance of red drum embryos to mechanical and chilling shocks depended on the embryo's stage of development. Gastrulae stage was more susceptible to mechanical shock than the other developmental stages. However, a different ontogenetic tolerance pattern for chilling shock was observed. It is suggested that the embryonic developmental stage needs be considered in future studies on the cryopreservation of fish embryos.

Several critical variables associated with successful cryopreservation of oyster embryos (Crassostrea gigas) were examined. These were 1) embryo developmental stage, 2) kind and concentration of cryoprotectant, 3) equilibration time, and 4) freezing rate. The percentage of survival was scored as the number of recovered embryos that swam actively 12 h after thawing and had developed into veliger stage. The oyster embryos became increasingly susceptible to the cryoprotectants as the concentration was increased and the equilibration time was lengthened. The stage of development appears to be a critical factor for survival of oyster embryos, with trochophore stage embryos more resistant than morula and gastrula stages embryos to cryoprotectant exposure and having better surviving after freezing. The optimum cryoprotectant concentration for the trochophore embryos differed markedly from the morula stage. Cryopreservation of fertilized eggs (2 to 8 cells) was unsuccessful. Varying degrees of success were achieved using gastrula- and trochophore-stage embryos. Maximum survival was obtained when trochophore embryos incubated in 10% propylene glycerol-artificial sea water were cooled at -2.5 degrees C/min to -30 degrees C and were then directly placed into liquid nitrogen. The results showed a clear effect of the stage of development on survival.

The effects of both osmolality and cation in the initiation of sperm motility were examined in yellowfin seabream, Acanthopagrus latus. Various factors involved in the cryopreservation of yellowfin seabream spermatozoa on motility are discussed. Extender containing only glucose proved to be a suitable medium for freezing yellowfin seabream spermatozoa to -196 degrees C. Glycerol seems to have a direct osmotic effect on yellowfin seabream sperm cells, and it induced sperm motility before freezing and during thawing. However, this exhausted the energy needed for sperm motility for fertilization. Dimethyl sulfoxide (DMSO) proved superior to ethylene glycerol, propylene glycerol, glycerol and methanol as a cryoprotectant. Prolonged equilibration time had a detrimental effect on both prefreezing and post-thawing sperm motility. The estimated optimum freezing rate was in the range of -20 to -154 degrees C/min. More frozen-thawed than fresh spermatozoa are required to achieve comparable fertilization rates.

The ultrastructure of spermatogenesis and of the spermatozoon of Acanthopagrus schlegeli (Sparidae) are described. The testis is of the unrestricted type. Germ cells are surrounded by cyst cells. Spermiogenesis involves conspicuous modifications such as intracellular movements (diplosome and mitochondria migration, nuclear rotation, and depression) and structural changes (chromatin condensation, shape of mitochondria, and loss of cytoplasm). The mature spermatozoon has a spherical nucleus with a deep, axial nuclear fossa, and an unusual notch, shaped like a bow tie. The short midpiece contains four spherical mitochondria and encircles the basal body of the flagellum. It is concluded that the A. schlegeli spermatozoon is of a primitive type, but that it is characterized by a unique feature which may provide a useful systematic character.

Ultrastructurally the spermatozoon of Acanthopagrus schlegeli (Sparidae) has a spherical, homogeneously electron-dense nucleus with a deep axial nuclear fossa, and an unusual notch, shaped like a bowtie, in the nuclear region. The short midpiece contains four spherical mitochondria and encircles the basal body of the flagellum. It is concluded that the spermatozoon is of a primitive type, although it is characterized by several unique features which may provide useful systematic characters. © 1993 Wiley-Liss, Inc., (Copyright © 1993 Wiley-Liss, Inc.)

A single dose of LHRHa (D-Ala(6), des Gly(10)- LHRH-ethylamide; 100 mug/kg body weight); administered by intramuscular injection, effectively induced ovulation in the Atlantic croaker (Micropogonias undulatus ); 37 of 40 (92%) females were successfully hand-stripped and there was no mortality. A single dose of human chorionic gonadotropin (hCG; 500 IU/kg body weight) was successful in inducing oocyte hydration. However, only 16 of 30 (53%) females ovulated successfully and could be hand-stripped. Another 8 females became extremely bloated and died. Thus LHRHa was shown to be the more reliable method of inducing ovulation in the Atlantic croaker. The response interval between hormone treatment and ovulation at different water temperatures was also determined.

The spermatozoon of the Atlantic croaker (Micropogonias undulatus) is a primitive type in that it lacks an acrosome. The kidney-shaped head has a diameter of about 1.5 microns and is occupied by a granular and electron-dense nucleus. The short midpiece contains 3 spherical mitochondria and encircles the basal body of the flagellum but is separated from it. The flagellum consists of the typical 9 + 2 axoneme and surrounding plasma membrane but lacks a lateral ridge. Spermatozoa of Atlantic croaker diluted in either NaCl or sodium citrate solutions with or without DMSO were examined with the electron microscope before freezing in liquid nitrogen and after thawing. Damage following cryopreservation appeared to be greater to the mitochondria, plasma membrane, and 9 + 2 axoneme than to the nucleus. The incidence of postthaw damage in spermatozoa diluted with NaCl solutions containing DMSO was remarkably lower than that with either pure NaCl solutions, pure sodium citrate solutions, or sodium citrate solutions containing DMSO.