Chromosome set manipulation and sex control in common carp: a review

The development of techniques for production of gynogenetic, androgenetic, polyploid, and monosex progenies in common carp (Cyprinus carpio L.) is described from a chronological perspective. Gynogenetic progenies were obtained either by suppression of the second meiotic division in eggs (meiotic gynogenesis) or by suppression of the first mitotic division in haploid embryos (mitotic gynogenesis). As a rule, gynogenetic progenies of common carp were all-female, revealing female homogamety (females—XX, males—XY) in this species. Induced gynogenesis results in increased homozygosity; the rate of increase depends on the type of gynogenesis. Inbreeding coefficient (F) for one generation of meiotic gynogenesis in common carp is about 0.6, while diploids obtained by mitotic gynogenesis are homozygous for all genes (F = 1.0). Mitotic gynogenesis was used for production of clones in common carp. In androgenetic progenies of common carp, YY males were identified, that after crossing with normal females (XX) produced all-male progenies. Triploids of common carp are characterized by a significant reduction in gonad development (especially ovaries). However, the reduction in gonad development did not result in an increase of somatic growth rate of fish. The procedure for androgen treatment to induce phenotypic sex reversal in genotypic females (XX) was elaborated. All-female progenies of common carp were produced on a large scale by crossing normal females (XX) with hormonally sex-reversed males (XX). Rearing of all-female progenies in conditions when fish normally reach sexual maturity before reaching of market size increased production yield by 7–8%. In a few cases distant hybridization resulted in polyploidy of fish without application of any physical treatment. The ability of hybrid females between crucian carp (Carassius auratus) and common carp to produce diploid (with unreduced chromosome number) gametes resulted in opportunities to produce triploid and tetraploid hybrid progenies.     

Effects of Intraspecific Hybridization in Tilapia nilotica on Survival Under Ambient Winter Temperature in Alabama

Viability under ambient winter temperatures in Alabama was assessed for Auburn University-Egypt and Auburn University-Ivory Coast strains of Tilapia nilotica and their F₁, F₂, and four backcross hybrids. Egypt strain was more viable ( P = 0.05) than Ivory Coast strain. Observed mean viabilities of the F₁ hybrids were intermediate between those of the parental strains. Viabilities of the reciprocal F₁ hybrids were not significantly different. Viabilities of the reciprocal F₂ hybrids were not significantly different. Backcross hybrids that were produced by crossing F₁ hybrid females to Egypt strain had a greater observed mean viability than those that were produced by crossing F₁ hybrid females to Ivory Coast strain. Average viabilities of the parental strains, F₁, F₂, and backcross hybrids were not significantly different. Heterosis in F₁, F₂, and backcross hybrids was −1.39%,−0.82%, and −2.08%, respectively. Viability under ambient winter temperatures in Alabama in T. nilotica appears to be controlled by additive rather than by dominance effects, which means that selection rather than crossbreeding is the breeding technique that should be evaluated to improve this trait in T. nilotica .     

Inheritance and reliability of random amplified polymorphic DNA-markers in two consecutive generations of common carp (Cyprinus carpio L.)

Random amplified polymorphic DNA (RAPD) markers have been used in a variety of genetic studies in fisheries and aquaculture. Most population studies are performed without preliminary data demonstrating the Mendelian inheritance and reproducibility of RAPD markers. In this study, the inheritance and reproducibility of RAPD markers was examined in two consecutive generations of common carp, Cyprinus carpio L. Variability and segregation of RAPD markers were investigated in one F₁ progeny and three F₂ progenies. Seventy-four RAPD markers were generated by five primers using DNA extracted from the initial ornamental (koi) common carp female and wild-type colour common carp male. Fifty-five of these RAPD markers were transmitted to the F₁ progeny and the inheritance patterns were analysed. Twenty RAPD markers were fully reproducible and demonstrated dominant simple Mendelian inheritance patterns in two consecutive generations. Twenty-four RAPD markers were not reproducible in all progenies. Thirteen markers displayed inheritance ratios in the progenies that did not fit simple Mendelian inheritance patterns. Non-reproducibility of RAPD markers and distorted ratios may be caused by the absence of amplification, poor amplification or by the appearance of artefact bands. Random amplified polymorphic DNA markers with poor reproducibility and non-Mendelian inheritance can lead to misinterpretations of data in population studies, resulting in errors in the estimation of genetic diversity within and between individual populations. Therefore, it is recommended to first identify the set of reproducible RAPD markers that demonstrate Mendelian inheritance before application of the RAPD technique in population studies.     

Estimates of Additive Genetic Effects, Maternal Genetic Effects, Individual Heterosis, Maternal Heterosis, and Egg Cytoplasmic Effects for Growth in Tilapia nilotica

Lengths and weights of Auburn University-Egypt and Auburn University-Ivory Coast strains of Tilapia nilotica and their F₁ F₂, and backcross hybrids were analyzed by multiple regression to estimate strain additive genetic effects, strain maternal genetic effects, individual heterosis (specific combining ability), maternal heterosis, and strain egg cytoplasmic effects for growth to determine why heterosis of the F₂ and backcross hybrids was greater than that of the F_I hybrids. Individual heterosis and maternal heterosis made the greatest contributions to growth. The F₂ and backcross hybrids were larger than the F₁ hybrids because of maternal heterosis. Regression coefficients were used to predict growth for any possible hybrid combination. Egypt ♀× Ivory Coast ♂ F_I hybrid females backcrossed to Egypt males and Egypt ♀× Ivory Coast ♂ F₂ hybrids were the largest groups evaluated in this study and were also predicted to have faster growth rates than any other possible Egypt-Ivory Coast hybrid.     

Variability in microsatellite DNA markers in gynogenetic and backcross progenies obtained from ornamental (koi) carp (Cyprinus carpio L.) × goldfish (Carassius auratus L.) hybrid females

Inheritance and segregation at five microsatellite loci were studied in diploid gynogenetic and triploid backcross progenies obtained from koi × goldfish hybrid females, which produce diploid eggs. Gynogenetic and backcross progenies were obtained from three individual hybrid females by inseminating eggs with genetically inactivated and intact sperm of parental species respectively; no shock treatments were applied to the early embryos. Complete absence of paternally specific alleles at all investigated microsatellite loci has proven successful genetic inactivation of spermatozoa by irradiation and confirmed gynogenetic origin of progenies. Genotypic segregations at microsatellite loci showed almost complete homogeneity of gynogenetic progenies and their identity to female parents. These results correspond with previous cytogenetic data on the occurrence of premeiotic endomitosis in hybrid females producing diploid eggs. Fish from triploid backcross progenies had genotypes resulting from combination of entire diploid female genome and haploid genome from male.     

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

The purpose of this study was to investigate the reproductive ability of second generation (F₂) koi (Cyprinus carpio L.) × goldfish (Carassius auratus L.) hybrids. Only diploid F₂ males and females were fertile and used in crosses. A significant increase was recorded in male fertility in F₂ versus F₁. In contrast with an earlier study in which only one fertile F₁ male was found, about 20% of F₂ males produced sperm. The observed reproductive ability of F₂ hybrids was similar to that demonstrated by the only fertile F₁ male and F₁ females. F₂ males produced diploid spermatozoa and generated triploids when crossed with koi females. All triploid fish in these progenies were males indicating that F₂ males had a sex chromosome constitution of XY. F₂ females produced diploid eggs and generated mostly triploids when crossed with koi males. In progenies obtained by crosses of F₂ males with F₁ and F₂ females, most of the surviving juveniles (63%–100%) were diploid; a minority of juveniles were aneuploid (ploidy ranged from 2.1n to 3.6n). Diploid fish in these progenies were presumably the result of spontaneous androgenesis and gynogenesis, by the same mechanisms observed earlier in progenies obtained by crossing the F₁ male with F₁ females.     

Golden Shiner Notemigonus crysoleucas× Rudd Scardinius erythrophthalmus Hybrids: Gonad Histology and Spawning Trials

Hybrid fish were produced in ponds by crossing golden shiners × rudd ( Notemigonus crysoleucas × Scardinius erythrophthalmus ) and rudd × golden shiners. Histologically, testes from the hybrids contained spermatocytes that resembled those of the parental species, but no sperm were observed. Lack of sperm in hybrids could have been a seasonal effect. The ovaries of both types of hybrids contained variable numbers of morphologically normal, vitellogenic oocytes and numerous abnormal oocytes in the chromatin nucleolar stage. Abnormal oocytes were necrotic or had irregularly shaped nuclei and clumped chromatin. Many oocyte nests contained infiltrating macrophnges. Spawning trials with F₁ hybrids did not result in spawning behavior or production of F₂ generation fish. Colden shiner × rudd hybrids are of reduced fecundity, but sterility was not conclusively demonstrated.     

Survival,growth and fertility of gynogenetic diploids induced in the cyprinid loach, Misgurnus anguillicaudatus

Gynogenetic diploids were induced in the loach, Misgurnus anguillicaudatus, by applying cold shock to the egg after fertilization with spermatozoa genetically inactivated by ultraviolet-ray irradiation, to examine their survival, growth and fertility. Eggs were obtained from orange phenotype loach (homozygous for a recessive demelanogenesis gene). Spermatozoa were obtained from black phenotype loach (homozygous for a dominant melanogenesis gene), and from common carp, Cyprinus carpio. All hybrids between loach and carp were inviably abnormal.Eggs fertilized with loach or carp spermatozoa irradiated at doses of 6000 and 12000 erg/mm ² and then treated with cold water (1°C for one hour 4 and 5 min after fertilization developed into embryos with 61–78% survival. About 70–87% of newly-hatched fry were normal in appearance. Gynogenetic fish induced by both loach and carp spermatozoa showed lower rates of survival and growth than did the normal diploid controls. All gynogenetic fish from carp spermatozoa were female and 94.9% of the gynogenetic fish from loach spermatozoa were female. The second gynogenetic offsprings were produced from females selected from both gynogenetic groups.     

Production of meiotic gynogenetic and triploid sea bass, Dicentrarchus labrax L.: 1. Performances, maturation and carcass quality

Meiotic gynogenetic and triploid sea bass were produced by pressure shocks according to a previously published protocol. Pressure-treated groups did not survive as well as controls during early development and larval rearing. Performances, sexual maturation and carcass quality were examined over a period of 34–45 months. At the age of 34 months, growth of the gynogenetic fish was comparable to that of the control but inferior in the triploid fish. A predominance of male fish was found within the triploid groups, while diploid and meiotic gynogenetic fish showed equal proportions of the sexes. Gonadal maturation in triploid fish was significantly impaired, particularly in the females that showed rudimentary ovaries. Triploid males exhibited primary maturation but proved to be gametically sterile. Pressure-induced triploids did not grow as well as diploids, but these results might be ascribed to specific on-growing conditions (communal rearing). The performance of gynogenetic sea bass was comparable to that of control. The superiority of diploid fish over their triploid counterparts was confirmed during the final growing period and more clearly so in females. Performances of triploids varied according to their maternal origin. Overall, striking qualitative differences between diploid and triploid fish were found at the age of 34 and 45 months, although the results varied in a gender-specific manner. A strong maternal effect was also observed. The potential advantages of triploid sea bass for aquaculture purposes are discussed.     

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.     

Estimates of additive genetic effects, maternal effects, specific combining ability, maternal heterosis, and egg cytoplasm effects for cold tolerance in Oreochromis niloticus (L.)

Abstract. Cold tolerances of Auburn University-Egypt and Auburn University-Ivory Coast strains of Oreochromis niloticus (L.) and their F_1. F₂, and backcross hybrids were analysed by multiple regression to estimate strain additive genetic effects, strain maternal effects, specific combining ability (individual heterosis), maternal heterosis, and strain egg cytoplasm effects for cold tolerance. The Egypt strain was more cold tolerant than the Ivory Coast strain. Cold tolerance of F₁ hybrids was intermediate between those of the parental strains. Heterosis for cold tolerances of F₁, F₂, and backcross hybrids was -1.39%, -0.83%, and -2.08% respectively. Additive genetic effects had the greatest effect on cold tolerance; Egypt genes for cold tolerance were superior to Ivory Coast genes for cold tolerance. Regressions for specific combining ability and maternal heterosis were small and were not different from zero, which explains why F₁, F₂, and backcross hybrids exhibited no hybrid vigour for cold tolerance. Cold tolerance in O. niloticus is controlled by additive rather than by dominance genetic variance; consequently, selection is the breeding technique that should be used to maximize cold tolerance. Regressions were used to predict cold tolerances of all possible Egypt-Ivory Coast hybrids. Predicted cold tolerances suggested that no possible Egypt-Ivory Coast hybrid would be more cold tolerant than the Egypt strain.     

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。     

The effect of ploidy and temporal changes in the biochemical profile of gibel carp (Carassius gibelio): a cyprinid fish species with dual reproductive strategies

The basic biochemical profile of gibel carp (Carassius gibelio), diploid–triploid cyprinid species complex with dual reproduction modes, was investigated. The aim of the study was to test for the effect of fish group (which combines the effects of ploidy and sex) and temporal changes on indicators of the biochemical profile of blood in gibel carp. We showed that the basic biochemical profile of blood is significantly influenced by season and fish group. Triploid gynogenetic females had a better condition measured by total protein concentration than sexual diploids. The higher concentrations of triacylglycerols and cholesterol in triploid females when compared to diploids indicate a higher metabolic rate and higher energy intake during the whole year, which may represent a disadvantage for triploids. Differences in the concentrations of blood biochemical analytes between triploid females and diploids suggest a potential difference in the regulation of a wide range of physiological and biochemical processes in which these analytes participate. The biochemical profiles of two forms of C. gibelio could at least partially explain the changes of character of its naturally occurring populations.     

Isoenzymatic changes in grass carp, Ctenopharyngodon idellus Cuvier & Valenciennes, affected with haemorrhagic disease

Abstract. Electrophoretic analysis of six types of tissue (serum, erythrocyte, liver, kidney, muscle and eye) and seven different isoenzymes (LDH, MDH, Est, ADH, IDH, α-GPDH and SOD) from both healthy grass carp, Ctenopharyngodon idellus Cuvier & Valenciennes, and carp affected by haemorrhagic disease was performed. It was found that: (1) Enzymes from diseased grass carp showed the most drastic changes within each tissue. The absolute activity of LDH in serum from the diseased fish increased to approximately twice that of the healthy fish; whereas the relative activity of A₄ and A₃B₁, the main components of A sub-base in both serum and erythrocytes, was accelerated. (2) The isoenzymic changes in the control groups, diseased fish, artificially infected fish and healthy fish from diseased ponds indicated that biochemical changes occurred in the tissues before pathological change.     

Increasing the genetic uniformity of bighead carp [Aristichthys nobilis (Richardson)] by means of spontaneous diploidization of gynogenetically activated eggs

Three groups of gynogenetic diploid bighead carp were successfully obtained by means of artificial gynogenesis. The activation rates of gynogenesis varied from 75.9% to 98.8%, and the frequency of spontaneous diploidization was around 0.4%. Over 2000 normally gynogenetic diploid fry were obtained in three gynogenetic groups. The haploid karyotype consisted of nine metacentric, 12 submetacentric, three subtelocentric chromosomes and 45 arms. The chromosome number was 48 from gynogenetic diploid. The results showed that the genetic material of offspring was maternal. The aneuploid hybrid embryos of bighead carp and Xingguo red common carp with chromosome numbers ranging from 28 to 73 did not survive post hatch, likely the result of incompatibility between the nucleus and the cytoplasm of two parents. Sixty RAPD primers from three groups were used for total DNA amplification of gynogenetic offspring, maternal and ‘paternal’ fish. A total of 451 bands were amplified from three kinds of samples above. From maternal bighead carp, 256 bands were amplified; however, there were 251 shared bands between maternal and gynogenetic bighead carp. From artificial gynogenetic offspring, two ‘paternal’ DNA segments without an expression function were found. An UPGMA tree showed that gynogenetic offspring were closely clustered and the genetic identity among them was very high (0.956).     

Verification of mitotic gynogenesis in ornamental (koi) carp (Cyprinus carpio L.) using microsatellite DNA markers

The Japanese ornamental (koi) carp is a popular decorative fish all over the world. In koi, clones have not yet been obtained, although production of fish with identical colour patterns could be of commercial interest. Mitotic gynogenetic progenies are essential for subsequent production of clones in fish. However, resulting late‐shocked progenies may be contaminated with meiotic gynogens from spontaneous suppression of the second meiotic division in eggs. In this study, microsatellite DNA markers were used to confirm mitotic gynogenetic origin of obtained late‐shocked progenies. Recombination rate (y) and mapping distance relative to centromere (M‐C) of 10 microsatellite loci were determined based on percentage of heterozygotes in meiotic gynogenetic progenies. The range of y varied from 0.01 to 0.96 and the M‐C map ranged from 0.5 to 48 cM. The mean value of y over the 10 loci was 0.481. Six loci, which had y 0.47 and higher, were used as markers in two late‐shocked gynogenetic progenies. Complete homozygosity was revealed at all six microsatellite loci indicating mitotic gynogenetic origin of analysed progenies.     

三倍体湘云鲫及其亲本线粒体DNA的比较研究

用人工选育的异源四倍体鲫鲤（♂）与白鲫（♀）杂交获得具有明显生长优势的三倍体湘云鲫。采用差速离心和核酸酶处理等方法，从三倍体、白鲫和异源四倍体鲫鲤肝组织中提取线粒体DNA，并用9种限制性内切酶进行单酶酶切分析。经琼脂糖凝胶电泳后计算出各酶切片段的大小，测得三倍体、白鲫和异源四倍体鲫鲤mtDNA的分子大小分别为16．24kb、16．60kb和16．20kb。根据各单倍型间的酶切片段共享度，估算出3个群体间的遗传距离，说明了mtDNA母系遗传的特性。     

Changes in enzymatic and structural properties of grass carp fast skeletal myosin induced by the laboratory-conditioned thermal acclimation and seasonal acclimatization

ABSTRACT:   Myosins were prepared from fast skeletal muscles of grass carp thermally acclimated to 10, 20 and 30°C in the laboratory as well as from those seasonally acclimatized and collected in January (winter) 2003 and May (spring), August (summer) and November (autumn) 2002. The maximal initial velocities ( V _max) of actin-activated Mg²⁺-ATPase activity for myosins from the 10°C-acclimated and winter grass carp were 1.7–1.8-fold as high as those from the 30°C-acclimated and summer fish. The inactivation rate constant ( K _D) of Ca²⁺-ATPase for myosin from the 10°C-acclimated grass carp was three to fourfold higher than those for myosins from the fish acclimated to 20°C and 30°C, whereas myosin from winter grass carp was about sevenfold as high as that for myosin from summer fish. Myosins from spring and autumn fish showed K _D values comparable to those of the fish acclimated to 30°C and 10°C, respectively. In differential scanning calorimetry analysis, the transition temperature ( T _m) was observed near 38°C and 45–46°C with most myosins. However, the lowest T _m at 32–33°C was given as one of the major endotherms in myosins from the 10°C-acclimated, autumn and winter fish. These responses of grass carp to changed environmental temperatures were almost similar to those for common carp reported previously.     

雌核发育二倍体鲫鲤及其他倍性鱼cdc2基因cDNA全序列克隆及表达分析

为研究雌核发育二倍体鲫鲤产生二倍体卵子的分子机制,实验采用PCR和cDNA末端快速分离法,克隆获得了雌核发育二倍体鲫鲤第三代(G3)、二倍体红鲫、三倍体湘云鲫和四倍体鲫鲤的细胞周期相关基因——cdc2基因cDNA全序列.结果显示,4种不同倍性鱼cdc2基因均编码含有302个氨基酸蛋白,而且编码的蛋白都含有与其他CDK激酶相当保守的序列PSTAVRE;同源性分析发现,4种鱼cdc2基因编码的氨基酸序列之间的相似度大于97.6％,说明Cdc2蛋白在这4种不同倍性鱼中具有高度保守性.采用实时荧光定量PCR(real-time PCR)对cdc2基因在G3、二倍体红鲫、三倍体湘云鲫及四倍体鲫鲤早期卵巢中的表达进行分析,结果发现,G3cdc2基因比普通二倍体红鲫和三倍体湘云鲫表达要高,比四倍体鲫鲤的表达水平低.该研究从分子水平证明了G3早期性腺中存在着大量的多倍体卵原细胞.同时,研究表明,cdc2基因在雌核发育二倍体鲫鲤早期卵巢的高表达暗示着G3多次进入S期却不经历M期导致二倍体配子的产生.