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.     

Inter‐specific hybridization between Black Sea trout (Salmo labrax Pallas, 1814) and rainbow trout (Oncorhynchus mykiss Walbaum, 1792)

Thermal shock‐induced triploid and unshocked control hybrids between rainbow trout (Oncorhynchus mykiss) and Black Sea trout (Salmo labrax) and their parental species were produced under hatchery condition by using heat shocks. Triploidization reduced eyed egg rate and alevin yield in all groups. Low survival rate was observed in both shock‐induced triploid hybrid and non‐shock‐induced control hybrids. Although hybrids demonstrated low body weight during the first feeding stage, they reached higher body weight by day 200 when compared with Black Sea trout and rainbow trout. A higher specific growth rate was calculated as 3.60 in the triploid hybrid groups, 1.41 in the triploid Black Sea trout groups and 2.27 in the triploid rainbow trout groups between days 110 and 200. A lower condition factor was determined in the hybrid than in the diploid parental species. A negative value of mid‐parent heterosis (MPH) performance was deected for condition factor, and a favourable MPH was detected for specific growth rate and weight in hybrids.     

Characterization of triploid hybrid groupers from interspecies hybridization (Epinephelus coioides ♀ × Epinephelus lanceolatus ♂)

Interspecies hybridization is widely used in aquaculture as a beneficial strategy. Diploid and triploid hybrids have been detected from the interspecies hybridization of Epinephelus coioides ♀ × Epinephelus lanceolatus ♂. This is the first report of triploidization through hybridization in grouper. Confirmation has been obtained through flow cytometry, karyotyping and erythrocyte nuclei measurement. The chromosome numbers of E. coioides, E. lanceolatus, diploid hybrid grouper are 48 and triploid hybrid grouper are 72. Measurements of erythrocyte nuclei indicate that triploid fish have a larger nuclear surface than the diploid groupers, and the average ratio of triploid to diploid surface area is 1.59. During the first 1.5 years, triploid hybrid groupers grow faster than diploid hybrid groupers or either parent species. The average growth rate of triploid hybrids is 1.61 times greater than that of diploid hybrids at 6 months of age and 1.43 times greater at 18 months of age. The triploid hybrid groupers are inferior in gonadal development, with no primary‐growth‐stage oocytes appearing in the gonads at 18 months of age. Morphological studies indicate that triploid hybrid groupers have distinctive differences in snout length, eye diameter, body trunk shape, and tail shape development compared with diploid hybrid groupers. Triploid hybrid groupers have an advantage in growth ability, and artificial breeding of triploid groupers might be of great potential use in the grouper aquaculture industry.     

Accelerated Growth, Gigantism and Likely Sterility in Autotransgenic Triploid Mud Loach Misgurnus mizolepis

Triploidy was induced in two selected lines of autotransgenic mud Ioach Misgurnus mizolepis containing no heterospecific genetic material. Cytological evaluations, including cellular DNA content, chromosome count, and erythrocyte measurement, proved the successful induction of triploidy. Patterns of Southern blot hybridization and tissue distribution of GH mRNA in autotransgenic triploids were similar to those of diploid autotransgenics. The autotransgenic triploids also displayed growth acceleration 22–25 times that of nontransgenic diploids, although the acceleration was relatively modest when compared to their diploid transgenic counterparts (more than a 30-fold increase). Significantly improved feed conversion was observed in both autotransgenic diploids and triploids, ranging from 1.8 to 2.0 times that in nontransgenics. Autotransgenic triploids were sterile at gonadal level, evidenced by significantly retarded and abnormal gametogenesis. With the persistence of accelerated growth and enhanced feed conversion efficiency, the autotransgenic triploid mud loach may offer economic benefits to aquaculture. Total absence of heterospecific genetic material and sterility may also allay public concern regarding food safety and environmental risk of crossbreeding between transgenics and conspecific populations.     

Genetic characterization, morphometrics and gonad development of induced interspecific hybrids between yellowtail flounder, Pleuronectes ferrugineus (Storer) and winter flounder, Pleuronectes americanus (Walbaum)

Viable interspecific hybrids between yellowtail flounder (Pleuronectes ferrugineus, Storer) and winter flounder (Pleuronectes americanus, Walbaum) were produced by artificial insemination of yellowtail flounder eggs with winter flounder sperm. However, mean fertilization rate, hatching success and early survival up to 3 weeks post hatch were significantly lower than those of parental pure cross controls (P < 0.01). Overall, cytogenetic traits (karyological analysis and estimation of cellular DNA contents using flow cytometry) of hybrid flounder were intermediate between the two parental species. Microsatellite assay was used to distinguish the parental genomes in the hybrids; in most cases, one allele was specific to each of the parents. Morphometrics assessed by body proportions indicated that hybrids generally displayed a morphology intermediate between the maternal and paternal species. Interspecific hybrids exhibited abnormal and retarded gonad development in both sexes based on histological analysis of gonads from adult fish. The sterility of the hybrids presents a significant advantage for their use in aquaculture, as potential escapees would not be capable of reproducing in the wild and contaminating natural stocks.     

Induced diploid gynogenesis and polyploidy in crucian carp, Carassius auratus gibelio (Bloch), x common carp, Cyprinus carpio L., hybrids.

Abstract. Results of a 16 year (1977–1992) study of hybrids obtained from crossing two cyprinid fish, crucian carp, Carassius auratus gibelio (Bloch) (bisexual form), and common carp, Cyprinus carpio L., are presented. The ability to produce diploid eggs in F₁ females (resulting from chromosome endoreduplication in early oogenesis) was responsible for the high yield of spontaneous diploids in gynogenetic progenies of F₁ females and for triploidy in hybrids from back-crosses of F₁ females with males of the parent species. Different types of hybrids, including diploid and triploid gynogenetic, tetraploid (amphidiploid) and androgenetic progenies, were obtained. Diploid and some triploid hybrids (females only) were reproduced inter se by induced gynogenesis in several subsequent gynogenetic generations. Amphidiploid hybrids were obtained by crossing triploid F₈ females with diploid common carp males or by crossing diploid gynogenetic females with diploid inverted hybrid males producing diploid sperm. Results of cytogenetic investigations and fish culture properties in hybrids are presented. The scientific and practical significance of the data obtained are discussed.     

Ploidy polymorphism and morphological variation among reciprocal hybrids of Pseudosciaena crocea (♀) × Miichthys miiuy (♂)

Diploid and triploid hybrids of large yellow croaker (Pseudosciaena crocea, Pc) and Miiuy croaker (Miichthys miiuy, Mm) were produced by interspecific hybridization. The result showed that Pc (♀) and Mm (♂) can be successfully crossed and hybrid offspring of normal hatch rate were produced. In contrast, a lower fertilize and hatch rate were produced for hybrids of Pc (♂) and Mm (♀), and the offspring died day 19 after hatching. To confirm the ploidy of hybrid offspring, we examined the karyotypes of offspring. The result of DNA relative content investigation and genetic relationship analysis demonstrated that the major hybrid offspring were hybrid diploid. In addition, a small number of hybrid were triploid and gynogenesis diploid. Morphological analysis based on the measurable traits data indicated the success of the interspecific crosses. The natural gynogenetic diploid large yellow croaker (PP), diploid hybrids (PM) and triploid hybrids (PPM) were closer to maternal parent (Pc) than paternal parent (Mm). This study provided genetic evidences of the chromosome, DNA content and morphological levels to support the successful establishment of the polyploidy hybrids of Pc (♀) and Mm (♂).     

Farming evaluation of the brownbow triploid hybrid (<Emphasis Type="Italic">Oncorhynchus mykiss</Emphasis><Emphasis Type="Italic">Salmo trutta</Emphasis>)

Triploid hybrids between female rainbow trout (Oncorhynchus mykiss Walbaum) and male brown trout (Salmo trutta L.) were tested for farming performances, with reference to parental species. The main drawback of hybrids lay in embryonic and larval mortalities, amounting to 60% on average, and displaying a large variability between spawns. Further survival was inferior to that of diploid, but similar to that of triploid rainbow trout. Hybrid body weight was intermediate between weights of rainbow and brown trout of the same age, mainly as a consequence of differences in precocious growth. Analysis of relative growth rates from 6 to 18 months showed that hybrids were surpassed by rainbow controls in common rearing, but not in separate rearing. Hybrid behaviour was similar to that of rainbow trout. These results are discussed in the scope of providing fisheries managers with original and sterile game fishes. An erratum to this article can be found at .     

Growth and Survival of Intrastrain and Interstrain Rainbow Trout (Oncorhynchus mykiss) Triploids1

Triploid rainbow trout exhibit improved survival and extended growth during sexual maturation, compared to their diploid counterparts. However, there have been few benefits demonstrated prior to sexual maturation. This study was undertaken to investigate the possibility of improving growth and survival parameters in triploids through interstrain crosses. Triploids were induced by heat shocking fertilized eggs from intra- and interstrain crosses of two rainbow trout strains. The four triploid groups and their diploid counterpart groups were reared to 233 days post-hatching and analyzed for growth and survival characteristics. Compared with diploids, triploids had significantly (P < 0.05) higher mortalities during the first 100 days post-fertilization, primarily just prior to and after hatching. However, during the remainder of the study triploids exhibited significantly (P < 0.05) lower mortalities than diploids. During the first 50 days of rearing all four triploid groups were significantly shorter and lighter than their diploid counterparts. The growth of the triploid groups later in the study varied considerably. At the conclusion of the rearing phase, one interstrain triploid group was significantly (P < 0.05) longer than its diploid counterpart, although not significantly heavier. The other triploid groups were either significantly smaller than, or equal to the diploids. Analysis of variance indicated that the growth of triploid rainbow trout was significantly affected by maternal strain effects. These results suggest that the use of specific strains and crosses may improve the growth of triploid rainbow trout.     

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.     

冷休克诱导泥鳅三倍体的研究

采用冷休克抑制第二极体排出的方法研究了泥鳅(Misgurnus anguillicaudatus)三倍体,并探讨了诱导泥鳅三倍体形成的最佳条件。结果显示:受精后3~5 min,用2℃冷水休克处理30 min,不仅可以得到较高的三倍化率,还可维持相当高的成活率。另外,本实验发现泥鳅间期细胞核Ag-NORs的最高数目与染色体组数目具有明显的对应关系,可以作为判断胚胎倍性的简单标准。     

Growth and reproductive performance of triploid and diploid blacklip abalone, Haliotis rubra (Leach, 1814)

Growth and reproduction of triploid and diploid blacklip abalone Haliotis rubra (Leach, 1814) were compared in a 30-month study. Triploidy was induced by inhibition of the second polar body formation using 6-dimethylaminopurine (6-DMAP) or cytochalasin B (CB). There were no significant differences in growth and survivorship between triploid and diploid abalone. However, triploid abalone had a more elongated shell and greater foot muscles than diploid abalone. A slightly curvilinear growth in shell length was conformed to all treatments. While diploid abalone had reached sexual maturity and spawned during the study, gonadal development and gamete maturation were abnormal in triploids. Female triploids lacked an apparent gonad at the macroscopic level but microscopic examination revealed that they had a thin layer of oogonia development. In contrast, male triploids were able to form similar-sized gonads to diploids during most of the reproductive period, but with brown-yellow discolouration and stalled gametogenesis at spermatocyte formation. Sex ratio of triploid abalone did not deviate from 1:1. With the onset of sexual maturation, growth and gonadal maturation occurred concurrently in diploid abalone, and there was no indication that growth of (diploid) abalone was reduced.     

Effects of vitamin E supplementation on growth,lipid peroxidation and fatty acid composition of Dojo loach (Misgurnus anguillicaudatus Cantor) fingerlings

A high mortality of cultured loach during their early development has constrained loach aquaculture industry in China. The objective of this study was to evaluate the effects of dietary vitamin E (VE) supplementation on growth performance, lipid peroxidation and fatty acid composition of loach Misgurnus anguillicaudatus fingerlings. Six test diets supplemented with 0 (0E), 50 (50E), 100 (100E), 150 (150E), 200 (200E) and 500 mg kg^?1 (500E) of dl‐α‐tocopheryl acetate were fed loach fingerlings (mean initial weight 24.0 ± 0.2 mg) in triplicate for 40 days. Results showed that the fish fed diet 100E and 200E had significantly higher body weight gains and specific growth ratios and significantly lower feed conversion ratios compared with fish fed diet 0E. With the increase in dietary VE level, whole‐body VE content was significantly increased (except 500E) and thiobarbituric acid reactive substances value was significantly reduced. Increasing dietary VE level increased the percentages of 20:4n‐6 and 20:5n‐3 in neutral lipid fraction, but decreased the percentages of 20:4n‐6 and 20:5n‐3 in polar lipid fraction. In conclusion, VE requirement of loach fingerlings for optimum growth and functionally preventing lipid peroxidation was 136.1 mg kg^?1 of dry diet under the present experimental conditions.     

Triploidization in rohu × mrigal hybrid and comparison of growth performance of triploid hybrid

The present study was aimed at the identification of treatment optima to induce triploidy in ‘Labeo rohita (rohu) × Cirrhinus cirrhosus (mrigal)’ hybrid using heat shock treatment. The eggs were exposed at four different temperature regimes viz., 38, 39, 40 and 41°C for 1–3 min, applied 3–5 min after fertilization. After 4 min of fertilization, heat shock treatments for 1 and 1.5 min durations were found the best inducing triploidy up to 100% and 96% respectively. Survival rates upto yolk sac absorption were found to be 73% and 71% in rohu and mrigal, 68% and 67% in the reciprocal diploid hybrids and 61% and 60% in the reciprocal triploid hybrids (RTH). Triploidy was confirmed by chromosome counting that revealed the diploid chromosome number of rohu and mrigal at 2n = 50 and in their triploid hybrid chromosome number was found to be 3n = 75. Growth rate of the RTH showed a significant difference (P < 0.05) from the single species and the diploid hybrids. Triploids also showed higher survival rate over the diploids.     

Effects of different dietary vitamin C supplementations on growth performance,mucus immune responses and antioxidant status of loach (Misgurnus anguillicaudatus Cantor) juveniles

A 60‐day feeding trial was conducted to determine the effects of different dietary vitamin C levels on growth performance, immune response and antioxidant capacity of loach juveniles. Six isonitrogenous (58.6% of crude protein), isoenergetic (17.5 kJ g^?1) practical diets supplemented with 0 (VC0), 100 (VC100), 200 (VC200), 500 (VC500), 1000 (VC1000) and 5000 mg kg^?1 (VC5000) of VC (35% ascorbic acid equivalent) were fed to fish (mean initial weight 0.11 ± 0.02 g) in triplicate. Results showed that fish fed VC0 diet had significantly lower body weight gain (BWG) and survival rate (SR). However, BWG and SR improved significantly in fish fed VC100 and VC200 diet respectively. Whole body ascorbic acid concentration increased with incremental dietary VC levels from 0 to 100 mg kg^?1. The activity of mucus alkaline phophatase was significantly increased by the dietary VC level. Incremental levels of VC significantly reduced activities of glutathione peroxidase (GPx) and catalase. Moreover, fish fed diets containing more than 100 mg kg^?1 VC significantly down‐regulated the superoxide dismutase and GPx mRNA expression in liver. Meanwhile, the expressions of liver heat shock protein (HSP70) and nuclear factor‐erythroid 2‐related‐2 (Nrf2) were affected by fish fed diets containing VC from 100 to 5000 mg kg^?1. In conclusion, VC requirement of loach juveniles for optimum growth and functionally preventing lipid peroxidation was more than 200 mg kg^?1 of diet. Moreover, high dose of VC supplementation did not show any detrimental effects on loach growth performance.     

Growth and feeding in juvenile triploid and diploid blacklip abalone, Haliotis rubra (Leach, 1814), at two temperatures

Growth and feeding of juvenile triploid and diploid blacklip abalone Haliotis rubra (Leach, 1814) were investigated at two temperatures of 17 and 21 °C over a 50‐day period. There were no differences in growth between triploid and diploid abalone as measured by shell length and body weight. Both triploid and diploid abalone increased in length but not in weight at 21 °C. Condition indices were similar for triploid abalone maintained at both temperatures; however, those for diploid abalone were significantly higher at 17 °C than at 21 °C. Food intake was significantly greater yet feed conversion efficiency was significantly lower in triploid than in diploid abalone. Both the feeding variables were independent of temperature. On average, diploid abalone were able to convert 1 g of dry food ingested to 0.58 g of body weight, but triploid abalone only 0.44 g.     

Reciprocal Hybrids among Diploid,Tetraploid Dojo Loach,Misgurnus anguillicaudatus,and Large‐scale Loach,Paramisgurnus dabryanus: Fertilization,Survival and Growth Performance

To determine the genotype with better traits for aquaculture practices of the loach, factorial crosses were made among diploid (D), tetraploid (T) dojo loach, Misgurnus anguillicaudatus, and large‐scale loach, Paramisgurnus dabryanus (P), producing DD, DT, DP, TD, TT, TP, PD, PT, and PP genotypes (female listed first). The growth performance and survival of different genotypes in culture were evaluated through a 48‐wk rearing trail. The average fertilization rate in genotypes with P. dabryanus as female parental was significantly higher than those of M. anguillicaudatus as female parental. The average fertilization level of sperm from P. dabryanus was about the same as diploid M. anguillicaudatus but significantly higher than tetraploid M. anguillicaudatus. The highest survival rate was found in DD group (52.77%) and PP group (50.31%) (P > 0.05). The intergenetic crosses between P. dabryanus and M. anguillicaudatus, especially for PD (19.05%), DP (17.89%), and PT (15.44%) groups, exhibited poor survival rates. Under the similar rearing conditions, PP group showed significant growth advantage. Growth rates of the interspecific hybrid progenies were found to be intermediate in comparison with the parental species. According to these results, the PP genotype is more suitable for aquaculture practice .     

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.     

Covariation between diploid and triploid progenies from common breeders in rainbow trout, Oncorhynchus mykiss (Walbaum)

The covariation between diploid and triploid progenies from common breeders was investigated in various progeny-testing experiments where either dams or sires were sampled from rainbow trout, Oncorhynchus mykiss (Walbaum), stocks. Triploidiza tion was found to frequently reduce the performance in the traits studied: body length and weight, growth, coefficient of condition and pyloric caeca number. Triploidization also generated significant interactions with the parental breeding value. These interactions were caused in part by the familial variance not being the same in triploids as in diploids, but also by actual ranking differences between diploid and triploid familial performances. However, the effect of these interactions was minor as compared with the amount of variation common to both ploidy levels (genetic correlations averaged 0.7–0.9). Therefore, selection of diploid breeders appeared efficient enough for improving triploid progeny, unless family selection methods including triploid progeny testing were preferred for other reasons. Lastly, it was observed that variances from maternal origin tended to be larger in triploids, whereas variances from paternal origin tended to be smaller, as compared with diploids. This point was discussed referring to the genetic make-up of triploids and in the scope of dams and sires for selective breeding.     

The occurrence of hypertetraploid and other unusual polyploid loaches Misgurnus anguillicaudatus among market specimens in Japan

Exotic animals can cause genetic contamination of indigenous species if they escape and reproduce in wild populations. The loach Misgurnus anguillicaudatus and species related to it have been imported into Japan for commercial use. We collected live loach specimens from the central wholesale market in Tokyo. For the 451 specimens collected, ploidy status was examined by DNA content flow cytometry, and polyploid loaches with triploid, tetraploid, and other higher ploidy ranges were detected. Hypertriploid and hypertetraploid individuals were easily detected by flow cytometry using standard eudiploid, eutriploid, and eutetraploid controls, and the reproductive capacities of these hyperpolyploid males were then examined. Sperm of hypertriploid males did not exhibit active progressive motility, and major populations of spermatozoa or spermatozoon-like cells were detected in the triploid and hexaploid ranges. Motile haploid spermatozoa were very rare in sperm from hypertriploid males. Therefore, hypertriploid males were sterile, whereas hypertetraploid males produced fertile hyperdiploid spermatozoa with active progressive motility after contact with ambient water. Viable progeny occurred in crosses between normal wild-type diploid females and hypertetraploid males, but androgenotes induced by the fertilization of UV-irradiated eggs with the sperm of hypertetraploid males were inviable hyperdiploids. Cytogenetic analyses of those androgenotes indicated that hypertetraploid males should produce hyperdiploid spermatozoa with 2n?=?54 (i.e., four supernumerary microchromosomes).