Studies on sperm of diploid and triploid tench, Tinca tinca (L.)

The tench Tinca tinca is an interesting fish from the viewpoint of polyploidy and related atypical reproduction aspects. Triploid tench were produced artificially. Studies of spermiation as well as of sperm motility and structure were performed on several triploid and diploid males simultaneously with individual experimental crosses with diploid females to define their reproductive capacities. The testes of triploids visually looked less developed in the most of cases with lower sperm production (0.05 cm³ sperm per male), GSI and weight of testes compared to diploids (0.58 cm³ sperm per male). Analysis of variance showed significant influence of ploidy level on the percentage of motile spermatozoa. Triploidy did not change percentage of live spermatozoa and velocity of spermatozoa at the first time of sperm movement. The study of sperm structure by scanning electron microscopy revealed that most sperm cells were of normal structure with some anomalies. Sperm heads of triploid and diploid males were mostly round-shaped, 1.86±0.2 and 1.6±0.18 μm in diameter. The midpiece of triploid spermatozoa was slightly narrower than that of diploid ones with typical cylindrical shape. Flow cytometry revealed sperm cells of triploids to be largely aneuploid (1.47 n) with high mosaic DNA, oscillating from haploid DNA content (1.0 n) to diploid DNA content (1.9 n). Experimental crosses between triploid males and diploid females revealed that these males were capable to stimulate effective development with relatively high level of fertilization and hatching rates from 0 to 70%. In conclusion, triploidization does not seem to guarantee sterility of tench.     

二倍体泥鳅与大鳞副泥鳅及杂交F1精子结构与活力

二倍体泥鳅与大鳞副泥鳅杂交能够顺利获得杂交后代,其正反交核型都为（2n=49）,但杂交后代精巢表现出明显的发育迟缓现象,杂交泥鳅自交与回交实验表明,其雄性不可育。为了解二倍体泥鳅和大鳞副泥鳅杂交F₁雄性不育的原因,实验通过计算机辅助精子活力分析系统（CASA）、光学显微镜、扫描电镜、透射电镜、流式细胞技术分别对二倍体泥鳅（DD）、大鳞副泥鳅（PP）、其正反杂交F₁（DP）和（PD） 4种泥鳅精子的形态结构与活力进行了分析。结果显示,激活30 s时,DD和PP的精子运动率分别为76.50%±0.70%和75.17%±8.60%,极显著高于DP （3.65%±1.75%）和PD （2.68%±0.63%）,且杂交泥鳅的精子的平均运动速度（VAP）、平均曲线速度（VCL）、平均直线运动速度（VSL）等运动参数也极显著低于DD和PP,说明杂交泥鳅精子的活力极其低下。应用流式细胞技术对4种泥鳅精巢内细胞进行倍性鉴定,发现DD和PP精巢内大多为1N精子,而DP和PD精巢内除了正常单倍体精子外,还有大量的2N和4N以及少量的8N细胞。通过扫描电镜和透射电镜...     

Reproductive ability of triploid ornamental (koi) carp (Cyprinus carpio L.) × goldfish (Carassius auratus L.) hybrids and characteristics of their offspring

The purpose of this study was to investigate reproductive ability of backcross triploid koi (Cyprinus carpio L.) × goldfish (Carassius auratus L.) hybrids. These triploids have been obtained by crossing of F₁ hybrid females producing diploid eggs with males of parental species. Triploid hybrid females, when crossed with goldfish or koi males, produced mostly aneuploid fish with ploidy range from approximately 2.2n–3.2n with a mean value 2.5n; some fish in crosses of triploid females with koi males were tetraploid (4.0n). Since analysed fish had in their genomes one haploid set from parental males, the data indicate that triploid hybrid females mostly produced aneuploid eggs with ploidy range from approximately 1.2n–2.2n and a mean ploidy around 1.5n while some eggs were triploid. Triploid hybrid males were completely sterile and have not released any sperm after hormonal injection. Despite their low viability, some aneuploid fish obtained from triploid hybrid females were raised in indoor recirculating systems until the age of 2 years and their reproductive ability has been evaluated. One aneuploid female with ploidy 2.1n produced larvae with ploidy range from 2.9n to 3.4n with a mean ploidy of 3.1n when crossed with a koi male; about 60% of obtained larvae had ploidy from 3.0n to 3.2n. These data indicate that this female produced mostly eggs with unreduced ploidy level.     

Genetic characterization of the progeny of a pair of the tetraploid silver crucian carp Carassius auratus langsdorfii

Silver crucian carp Carassius auratus langsdorfii comprises a diploid-polyploid complex in wild Japanese populations. Bisexually reproducing diploids are sympatrically distributed with gynogenetically developing triploids and tetraploids. Triploid and tetraploid males are very rare among Japanese silver crucian carp due to their gynogenetic reproduction. We examined the genetic characteristics of progeny that arose in a tank by natural spawning of a tetraploid silver crucian carp pair. The ploidy status of 120 samples randomly collected from these progeny was determined to be tetraploid by DNA content flow cytometry. DNA fingerprints from a random amplified polymorphic DNA assay indicated that almost all the progeny examined had genotypes identical to the maternal tetraploid female with no paternally derived fragments. Selected specimens’ cytogenetic analyses revealed that the progeny examined had tetraploid chromosome numbers, categorized into 40 metacentric, 80 submetacentric, and 80 subtelocentric or telocentric chromosomes, which were arranged into quartets and six supernumerary microchromosomes. Fluorescence in situ hybridization signals were detected in four homologous chromosomes in all analyzed metaphases prepared from diploid goldfish specimens. Contrary, tetraploid silver crucian carp gave eight rDNA signals. These results suggest that gynogenetic development in eggs spawned by tetraploid females should be triggered by tetraploid males’ homospecific sperm.     

Growth performance, morphometric traits and gonad development of induced reciprocal diploid and triploid hybrids between the mud loach (Misgurnus mizolepis Günther) and cyprinid loach (Misgurnus anguillicaudatus Cantor)

Two species of loach, the mud loach (Misgurnus mizolepis Günther) and the cyprinid loach (M. anguillicaudatus Cantor), are commercially important in Korea both for food and ceremonial purposes. The mud loach has superior potential for aquaculture in terms of growth, whereas the cyprinid loach has a more desirable body shape and colour. This study was conducted to produce reciprocal diploid and triploid hybrids and to evaluate their potential benefits in culture by examining growth performance, morphometrics and gonad development. Reciprocal diploid and triploid hybridization was performed by artificial insemination without or with the induction of triploidy. The successful formation of diploid or triploid karyogamy was verified by flow cytometric analysis. Body weights of induced reciprocal diploid hybrids were intermediate between those of the two parental species, i.e. hybrids were heavier than cyprinid loach but lighter than mud loach. In contrast, the growth performance of triploid hybrids was similar to that of their corresponding maternal parents. Diploid hybrids showed intermediate morphometric traits between the two species; however, the body proportions of triploid hybrids were more similar to those of the maternal species than the paternal species. Histological analyses indicated that reciprocal diploid hybrids of both sexes were able to reach maturity, as evidenced by the presence of mature oocytes or spermatozoa in the gonad tissues. However, triploid hybrids showed stringent sterility at the gonadic level; the sizes of ovaries and testes were much smaller, and gonad development was abnormal and significantly retarded.     

Structural abnormalities of common carp <Emphasis Type="Italic">Cyprinus carpio</Emphasis> spermatozoa

Spermatozoa of common carp Cyprinus carpio are typically consist of a primitive head without acrosome, a midpiece with several mitochondria, a centriolar complex (proximal and distal centriole), and one flagellum. During an evaluation of the motility of common carp spermatozoa, we found spermatozoa with more than one flagellum and/or “double head” in three different individuals. This may be related to abnormal spermatogenesis. Ultrastructure and physiological parameters of spermatozoa were examined using light microscopy (dark field with stroboscopic illumination), transmission and scanning electron microscopy, and flow cytometry. The recorded pictures and videos were evaluated using Olympus MicroImage software. All spermatozoa with more than one flagellum had a larger head and shorter flagella. They occasionally demonstrated several cytoplasmic channels separating the flagella from the midpiece. Each flagellum was based upon its own centriolar complex, with the connection of the flagellum to the head always at a constant angle. The flagella always consisted of nine peripheral pairs and one central doublet of microtubules. Sperm exhibited a relative DNA content similar to that found in sperm from normal males, with higher coefficients of variation. Although similar abnormalities have been found in livestock, where they were described as a defect in spermiogenesis, no comparable results have been reported in fish. The frequency at which these abnormalities occurs, the fertilization ability of males with defects in spermiogenesis, the influence of these abnormalities on progeny in terms of ploidy level, and the occurrence of deformities warrant further investigation.     

微卫星分离模式显示雄性三倍体鲫产生非整倍体精子

三倍体鲫(Carassius auratus)行天然雌核发育,其自然种群中却有较高比例的雄性个体,这些雄性个体的性腺发育正常,能产生有活力的精子。而且其精子的DNA含量约为体细胞的一半,显示三倍体鲫精子发生过程中可能经历了均等的减数分裂。流式细胞术虽然能够较准确地测定细胞群的平均DNA含量,但是却很难检测到单个精子中的个别染色体增减,要明确回答雄性三倍体鲫产生的精子是否为整倍体需要定量地检测单个精子的遗传组成。本研究用微卫星标记检测以雌性鲤(Cyprinus carpio)与雄性三倍体鲫为亲本构建的杂种家系的基因型,结果发现母本鲤的多态位点在子代中呈孟德尔分离,父本三倍体鲫具有三套鲫基因组,其等位基因在子代中呈随机分离。上述研究结果提示:三倍体鲫起源于二倍体鲫的同源加倍,而非二倍体鲫和鲤的种间杂交;三倍体鲫通过染色体的随机分离产生非整倍体的精子,其精子发生过程中没有均等的减数分裂。三倍体鲫行雌核发育生殖,却可能并非起源于种间杂交且群体中的雄性个体可育,因而是单性生殖鱼类中的一个特例。     

Aquaculture performance of triploid barfin flounder Verasper moseri

ABSTRACT:   To evaluate the aquaculture performance of triploid barfin flounder Verasper moseri , the sex ratio, maturation, growth and the relative proportion of body parts were examined. The sex ratio of triploids was similar to diploids under communal rearing conditions, but the proportion of female diploids was higher than that of triploids under separate rearing conditions. The gonadosomatic index of triploid females was very low even during the spawning season, and the ovaries were rudimentary. These results suggest that triploid barfin flounder females were sterile. In addition, triploid males produced a small quantity of milt containing very few spermatozoa with abnormal shapes. Spermatozoa obtained from triploids were aneuploidies. When normal eggs were fertilized with sperm from triploid males, no fry developed. These results suggest that triploid barfin flounder males were functionally sterile. Triploid males grew more slowly than diploid males, and triploid females showed similar or slower growth than diploid females, whether reared separately (23 months) or communally (35 months). The ratios of visceral weight to the edible parts for triploid males were similar to those for diploid males, but ratios for triploid females were higher than for diploid females during the spawning period. In conclusion, a significant improvement of growth was not found in triploid barfin flounders.     

Spawning of ocean pout (Macrozoarces americanus L.): evidence in favour of internal fertilization of eggs

Sperm physiology, in vivo artificial insemination and spawning of the ocean pout (Macrozoarces americanus L.), a marine bottom fish, were studied. Milt was collected from the reproductive tract of mature males by suction using a catheter. The uncontaminated milt, having a very low sperm concentration, contains highly motile spermatozoa and sperm motility was retained in vitro at 4 °C for at least 24 h in both seminal plasma and ovarian slime collected from the oviduct of pre-spawning females. Instead of activating sperm, dilution in sea water instantly immobilized the spermatozoa of ocean pout. Osmolarity and pH of ocean pout seminal plasma were in the ranges 365–406 mOsM and 7.2–7.5, respectively. A study of the ionic composition of ocean pout seminal plasma demonstrated the presence of various ions including Na⁺, K⁺, Ca²⁺, Mg²⁺, and Cl⁻, with a remarkably lower K⁺ concentration compared to that from other fish species. Since injections of milt containing motile sperm into the ovaries of pre-spawning females, which spawned in the absence of males, yielded fertilized ocean pout eggs, it is concluded that the ocean pout exhibits internal fertilization. The larvae hatched after 3 months of egg incubation in ambient sea water (9–10 °C). With proper timing of in vivo artificial insemination of mature females, fertilized ocean pout eggs can be obtained from fish reared in captivity.     

Fertility and ploidy of gametes of allodiploid and allotriploid loaches produced by diploid <Emphasis Type="Italic">Misgurnus anguillicaudatus</Emphasis> females and <Emphasis Type="Italic">Paramisgurnus dabryanus</Emphasis> males

Artificial and natural hybridization of dojo loach Misgurnus anguillicaudatus (2N = 50, DD for short) and large-scale loach Paramisgurnus dabryanus (2N = 48, PP for short) are well-grown. However, these hybrid loaches have not yet been examined for fertility and ploidy of gametes. Here, histological observations, artificial propagation, observations of embryonic development, larval morphology, and ploidy analyses were conducted to determine the fertility and ploidy of gametes of allodiploid (DP for short) and allotriploid (DDP for short) loaches, produced by DD females × PP males and induced from fertilized eggs of DD females × PP males by cold shock to prevent the second polar body release, respectively. The ovaries of DP and DDP included smaller number of eggs when compared with those of the control DD, while full-grown oocytes were observed. Testes of these two loaches were delayed-developed without spermatids or mature spermatozoa. Results obtained here showed that DP and DDP were fertility-weakened female and sterile male. Moreover, DP females and DDP females could, respectively, produce few viable haploid eggs and few viable haploid and diploid eggs. This study will provide valuable information for fish hybrid researches.     

Induction of viable gynogenetic progeny using eggs and UV-irradiated sperm from the Chinese tetraploid loach, Misgurnus anguillicaudatus

When eggs from the Chinese tetraploid loach that had 100 chromosomes were fertilized with UV-irradiated sperm, we obtained viable gynogenetic progeny without any additional treatment for the duplication of maternal chromosomes, which survived beyond first feeding towards adult stage of development. Gynogenetic progeny were determined to be diploid since they possessed 50 chromosomes, along with two chromosomes bearing nucleolar organizing regions (NORs), detected by silver nitrate staining (Ag-NORs), chromomycin-A₃ (CMA₃)-positive sites and fluorescence in situ hybridization (FISH) signals for rDNA loci. In contrast, when gynogens were induced using eggs from diploid loach fertilized by UV-irradiated sperm, but without chromosome doubling, we found that all resultant progeny were non-viable haploid gynogens with 25 chromosomes, along with one NOR-bearing chromosome detected by Ag-NORs, CMA₃ and FISH. These observations demonstrate the true genetic tetraploid nature of the Chinese loach possessing 100 chromosomes, and the potential use of this tetraploid as a source of functional diploid gametes for further ploidy manipulation experiments.     

Heat-shock-induced tetraploid and diploid/tetraploid mosaic in pond loach, Misgurnus anguillicaudatus

Tetraploid fish, which are considered as key resources of diploid gametes for further breeding and ploidy manipulation, can be artificially induced by inhibition of the mitotic cell division with hydrostatic pressure or temperature treatments. Although many attempts have been made to induce artificial tetraploid strains, successful establishment of viable and fertile tetraploid strains are rare. In pond loach, Misgurnus anguillicaudatus, natural tetraploid individuals are distributed in wild populations and diploid gametes from the tetraploid fish have been used for the induction of polyploid individuals, but artificially induced tetraploid strains have not been established yet. In the present study, we optimised starting timing of the heat-shock treatment (41 °C for 2 min) to inhibit a mitotic cell division in fertilised eggs of the normal diploid pond loach between 21 and 51 min after insemination at 20 °C. After the treatment, we observed external appearance of hatching larvae and flow cytometrically determined ploidy status of the resultant larvae. Although tetraploid and diploid/tetraploid mosaic larvae were obtained, the optimum timings for induction of tetraploidy varied amongst crosses. Various kinds of ploidy such as haploidy, diploidy, triploidy, pentaploidy, hexaploidy, aneuploidy and mosaic were detected in non-optimum heat-shock timings for tetraploidisation. Survivors, a tetraploid and a diploid/tetraploid mosaic male, matured at the age of 1-year-old, but they produced functional haploid spermatozoa.     

Exposure to 17α,20β-dihydroxy-4-pregnen-3-one changes seminal characteristics in Nile tilapia, Oreochromis niloticus

This experiment was conducted to evaluate the seminal characteristics of Nile tilapia males exposed to water‐borne 17α,20β‐dihydroxy‐4‐pregnen‐3‐one (17,20βP). Male Nile tilapia (Oreochromis niloticus L.) were exposed to the steroidal pre‐ovulatory pheromone 17,20βP, added to water at a concentration of 5×10^?9 M. The pheromone‐exposed males had higher sperm volume and concentration. In addition, the spermatozoa contained in the sperm had higher motility and the motility duration was longer than ethanol‐exposed males (control group). The percentage of live spermatozoa was not affected by the treatments. Our results suggest that this pheromone can improve sperm quality characteristics and could become a non‐invasive method for enhancing spawning in Nile tilapia.     

Retention of sperm motility in turbot, Scophthalmus maximus L.: the effects of time from activation, thermal shock and adenosine triphosphate levels

Abstract Four parameters were examined in order to define sperm quality in turbot Scophthalmus maximus L., sperm: (1) sperm motility, measured by direct counts of the number of active spermatozoa, expressed as % of total spermatozoa; (2) retention of motility after activation, measured by direct counts, 0–60min after activation, expressed as a % of the initial level of activity; (3) resistance to thermal stress, measured as change in retention of motility, and (4) adenosine phosphate (ATP) concentration, determined for samples of non-activated sperm. The proportion of motile spermatozoa at activation ranged from 34·8% to 97·6% (mean 76·3%) for the individual males tested. Turbot sperm retained on average 52% (range 27–90%) of its initial activity one hour after activation. Sperm samples which were stressed by cooling to –27°C retained only 8·6% (range 0–25%) of initial activity, compared to control samples which retained 49% (range 38–63%) of initial activity. The retention of motility after activation was not significantly related to the initial motility or the levels of ATP. Concentrations of ATP in turbot sperm (mean 0·46mg ATP/10⁶ spermatozoa, equivalent to 9·2nmol ATP/10⁸ spermatozoa) were comparable to those measured in mammals.     

Induction of triploidy in mud loach (Misgurnus mizolepis) and its effect on gonad development and growth

Triploidy was induced in mud loach (Misgurnus mizolepis) by cold shocking fertilized eggs 5 min post-fertilization at 2°C for 15 to 60 min. Best results were obtained when eggs were shocked for 60 min; 98% of fish examined in that treatment were triploids. Triploidy was confirmed by erythrocyte measurements and chromosome counts. Diploids had 48 chromosomes, while triploids had 72. Histological analysis of 9-month-old triploid ovaries showed an appreciable number of oocytes at the chromatin nucleolus stage with considerable interstitial tissue. However, diploids had well developed oocytes. Diploid testes from diploid males exhibited normal spermatids and spermatozoa, while a few were seen in triploid males. Growth rate was evaluated over a 9-month growth trial. Although male and female triploids were slightly heavier than their diploid counterparts from the third to the ninth month, their growth rates were not significantly different compared to their diploid controls.     

High fry production rates using post-thaw silver carp (Hypophthalmichthys molitrix) spermatozoa under farming conditions

Silver carp (Hypophthalmichthys molitrix) is not endemic to Cuba, and egg fertilization is totally artificial; males produce spermatozoa only between March and October. Cryopreservation of silver carp spermatozoa would reduce the number of males needed, minimize handling stress through less frequent stripping, and facilitate artificial propagation when eggs are available. The effects on motility and fry production from eggs fertilized with thawed sperm under farm-conditions were examined in this study. Five, seven and ten percent of dimethyl sulfoxide (DMSO), glycerol and methanol were tested as cryoprotectants. DMSO was a more suitable cryoprotectant than methanol or glycerol. The effect of equilibration time on the motility rate at 10% DMSO was evaluated. Hatching rates equal to the control (P>0.01) were obtained under farm conditions with frozen spermatozoa, stored even for a year in liquid nitrogen, with a final DMSO concentration of 10%. Cryopreservation offers a useful routine method for sperm storage and silver carp handling. To our knowledge, this is the first report on the production of fry from post-thaw silver carp spermatozoa under farming conditions.     

Characterization of Senegalese sole, Solea senegalensis, male broodstock in terms of sperm production and quality

Sperm quality and production have never been characterized in Solea senegalensis males. Reproduction in captivity in this species has been obtained mostly with wild-captured animals, because it is common that the F1 generation fails to reproduce. However, there is no information on sperm quality from both types of broodstocks. The aim of the present study was to characterize sperm production and to describe the profiles of spermiation in individual wild-captured males. Also, sperm quality and production were determined in two types of broodstocks established in our facilities; wild-captured and F1 individuals. The males were analyzed for their fluency and identified as fluent or non-fluent. The sperm volume, cell concentration, sperm production and motility were recorded from mid February until mid November in both broodstocks. Results showed that S. senegalensis males can produce motile sperm during all this period, with specific peaks of high spermiation and a high percentage of fluent males. This fact was observed in both male broodstocks. There was a large variability in terms of sperm profiles in males maintained under the same conditions. Sperm volume collected in this species was very small and ranged from 5 to 20 μl in F1 broodstock and 10 to 80 μl in wild-captured broodstock. Cell density ranged from 0.7 to 1.2 × 10⁹ spermatozoa/ml in F1 males to values of 1-2 × 10⁹ spermatozoa/ml for the wild-captured males. Sperm production (total spermatozoa per stripping) was also very low and ranged from 20 × 10⁶ spermatozoa for F1 broodstock to 40-60 × 10⁶ spermatozoa for wild-captured broodstock. Our results demonstrated that sperm production in this species is very low and variable according to the type of males. These results suggest that a previous selection of males according to their fluency, sperm production and provenience (wild-captured or F1) should be taken into account in the establishment of a S. senegalensis broodstock.     

Influence of photoperiod, frequency of stripping and presence of females on sperm output in turbot, Scophthalmus maximus (L.)

Abstract. The effect of four environmental conditions was investigated upon sperm output in turbot, Scophthalmus maximus (L.), submitted to three different rhythms of stripping. Males kept under a natural light cycle and under a 6-month contracted light programme released a similar sperm output in terms of total volume of semen produced per fish during the experimental period (4·9 ± 0·9ml), mean sperm concentration (29·4 ± 2·8 × 10⁹ spermatozoa/ml) and total sperm number (163·2 ± 40·5 × 10⁹ spermatozoa). Attempts to stimulate spermiation for a second time just after the end of the natural reproduction period resulted in the release of low sperm output (total volume of semen: 1·6 ± 0·4 ml; mean sperm motility: 2 min 36s ± 0 min 47s; mean sperm concentration: 47·6 ± 10·2 × 10⁹ spermatozoa/ml; total sperm number: 84·5 ± 25·3 × 10⁹ spermatozoa). Stripping frequency had no effect on total volume of semen, mean sperm motility and total sperm number. Monthly collection did not modify sperm samples in relation to stripping rank. However, decreasing volume, motility and sperm concentration were observed when males were stripped fortnightly and weekly. During the natural spawning period, the presence of females in the tank enhanced mean sperm motility (from 3 min 27s + 0 min 52s to 6 min 38s ± 1 min 38s).     

Pre-spawning water temperature affects sperm respiration and reactivation parameters in male carps

Concentration, ability to motility, motility during the second activation (reactivation), and endogenous respiration were studied in sperm from two experimental groups of carp males. Group 1 was maintained for 7 days at 15°C (cold water (CW) group), whereas the second group was subjected to a temperature of 20°C (warm water (WW) group) before sperm sampling. Reactivation were achieved after incubation of firstly activated sperm in media with osmotic pressure adjusted up to 300 mOsm*kg⁻¹ by increasing K⁺ concentration. Statistically significant reduction of spermatozoa concentration in CW samples versus WW (from 46.0 ± 12.5 (15°C) to 59.3 ± 7 10⁹ (20°C) spermatozoa /ml) have been observed. The sperm of the CW group required a significantly longer incubation time (37 min) under isotonic conditions to achieve a maximum percentage of potent motility at repeated activation than the WW group (23 min). After activation of sperm motility, an increase of respiration rate up to maximum level has been found, this level remained the same under condition of recovering the potential to repeated activation. During the sperm movement respiration rate, in CW group (6.1 nmolO₂/min/10⁹spermatozoa) and WW (3.9 nmolO₂/min/10⁹spermatozoa), was significant higher compared to nonactivated sperm (2.4 nmolO₂/min/10⁹spermatozoa for CW and 1.1 nmolO₂ /min/10⁹spermatozoa for WW). And keeping males for 7 days at 15°C increase the respiration rate of sperm.