Production of a high percentage of male offspring in growth-enhanced transgenic tilapia using Oreochromis aureus ZZ selected pseudofemales

Gene transfer has offered a new tool for the development of improved fish strains for aquaculture. Monosex fish populations could minimize the impact of genetically modified organisms in the environment. In Oreochromis aureus, the use of pseudofemale spawners (sex-reversed male with a female phenotype) is an alternative technique for producing genetically male tilapia offspring. O. aureus fry were treated with 17β-estradiol at 100 mg/kg of food for 45 days. We obtained 77.1% females and 45.9% in the control group. Females randomly taken from the treated group were crossed with normal males. Fry from pseudofemales producing more than 90% male progenies were submitted to 17 β-estradiol treatment to obtain F₂ pseudofemales. The results of the sex-reversal were low and variable ranging between 66.0 and 84.3% females. F₂ pseudofemales were crossed with transgenic males from the F70 line (O. aureus × O. urolepis hornorum) and non-transgenic (O. aureus) males. The sex ratio of progeny of F₂ pseudofemale deviated significantly (P < 0.01) in favor of males in the crosses with transgenic (90.2%) and non-transgenic (89.3%) males compared to the results observed with normal females (51.0 and 52.3%, respectively). The mean fry production with pseudofemales (per m²/day) was similar to the normal females in the crosses with transgenic and non-transgenic males. To our knowledge this is the first report on the production of a near monosex population in genetically modified fish.     

Age, growth, and reproduction of silky sharks, Carcharhinus falciformis, in northeastern Taiwan waters

To examine the age, growth and reproduction of silky sharks, Carcharhinus falciformis, in the waters off northeastern Taiwan, 469 specimens (213 females and 256 males) were collected from August 2000 to January 2002 at the Nanfanao fish market, northeastern Taiwan. The relationship between body weight (W) and total length (TL) for both sexes combined was expressed as: W = 2.92 × 10⁻⁶ TL^3.15 (n = 469, p < 0.01). The relationship between TL and vertebral centrum radius (R) for both sexes combined data was estimated as: TL = 25.979 + 18.197R (n = 250, p < 0.01). Growth bands (including translucent and opaque zones) in precaudal vertebrae formed once a year between December and January and were counted up to 11 and 14 for females and males, respectively. The von Bertalanffy growth function (VBGF) was used to model the observed length at age data. The sexes combined VBGF predicted an asymptotic length (L_∞) = 332.0 cm TL, growth coefficient (k) = 0.0838 year⁻¹, age at zero length (t₀) = −2.761 year (n = 250, p < 0.01). Size at 50% maturity for males was estimated to be 212.5 cm based on the logistic curve, which corresponded to 9.3 years. Females matured at 210–220 cm, which correspond to 9.2–10.2 years. The length at birth was estimated to be 63.5–75.5 cm TL. The number of embryos per litter was 8–10 and sex ratio of embryos was 1:1.     

Photobacterium damselae subsp. damselae, an emerging pathogen in Danish rainbow trout, Oncorhynchus mykiss (Walbaum), mariculture

A selection of 16 field isolates of Photobacterium damselae from marine rainbow trout farms in Denmark was subjected to phenotypic and genotypic characterization and pathogenicity to fish. All isolates belonged to the subspecies damselae , being positive for haemolysis, motility and urease. There were considerable differences in haemolytic properties, some isolates presenting a broad zone of haemolysis and others only a narrow zone. Pulsed-field gel electrophoresis revealed a high diversity indicating that P. damselae subsp. damselae is an opportunistic, not clonal pathogen in Danish marine rainbow trout. Virulence of the strains to rainbow trout was highly variable with LD₅₀ values ranging from 3.9 × 10³ to 1.5 × 10⁸ cfu at 20 °C. The virulence was significantly higher at 20 °C than at 13 °C. The strains with the strongest haemolytic properties were the most virulent suggesting a strong involvement of haemolysin in the pathogenesis. The pathological changes were consistent with a bacterial septicaemia and the haemorrhages were more pronounced than for most other bacterial infections.     

Total calcium and inorganic phosphate in the blood plasma of farmed rainbow trout, Oncorhynchus mykiss

Reference (physiological) value intervals determined by the lower 2.5% and upper 97.5% quantiles were calculated for total calcium (Ca _t ) and inorganic phosphate (P) in farmed rainbow trout, Oncorhynchus mykiss , at an age of 10–12 months in raceway culture. Total calcium levels were significantly ( P ≤0.0009) greater in males (2.63–4 mmol L⁻¹, n =47, mean weight 359±142 g) than in immature females (2.48–3.97 mmol L⁻¹, n =410, mean weight 400±145 g). The reference range for P in immature females and males was 3.09–9.32 mmol L⁻¹ ( n =467, mean weight 401±144 g). The distribution and density of the quantiles in the tested reference group were made possible by the use of histograms, which showed normal distributions for Ca _t in males but not for Ca _t and P in immature females and males, in which a sinistral asymmetry was found.     

Genetic contribution of parents in sex determination of honmoroko Gnathopogon caerulescens

The honmoroko has been inferred to have an XX/XY sex determination system, but the parental genome can also affect the sex ratio of the offspring. The extent of parental effects on sex determination was examined by checking the sex ratios of F1 and F2 gynogenetic diploids and control diploids. Eleven gynogenetic broods from different females consisted of all or nearly all females, but eight broods showed a variable proportion of males (<50 %). One second-generation brood of gynogenetic diploids consisted wholly of females, but others produced some males. In crosses with a control diploid female, four males from a high-percentage male brood of gynogenetic diploids produced offspring with a balanced sex ratio. Sib-mating between a gynogenetic female and three gynogenetic males from the brood produced predominantly male progeny. These results suggest that there are at least four possible genotypes: genotypic female (XX), phenotypic female carrying a silent Y chromosome, genotypic male (XY), and genotypic supermale (YY). These inferences suggest that this fish has an XY system but a relatively high proportion of females possess a mutated, silent Y chromosome which does not lead to testis formation.     

Sex reversal in salmonid culture: Part II. The progeny of sex-reversed rainbow trout

A sample of the all-male population of rainbow trout resulting from the dietary administration of 17α-methyltestosterone at a concentration of 3 mg/kg of food to juvenile fish during the 90-day period following “swim up” was grown to sexual maturity. It was observed that in many specimens there were deformations of the reproductive system which prevented the normal expression of milt although spermatozoa from the testes were viable.Sperm specimens removed surgically or normally expressed from mature fish were separately used to fertilise the mixed ova from untreated females. All the progeny of one such pairing were females. This indicates that in the rainbow trout it is the female sex which is homogametic.Samples of the all-female population which resulted from the dietary administration of 17β-oestradiol at a concentration of 20 mg/kg of food to rainbow trout fry during the 56-day period following “swim up” were also grown to maturity. The ova stripped from these fish were separately fertilised with mixed milt expressed from normal males. The progeny of two of five such pairings had a combined male to female ratio of 2.2, indicating that in rainbow trout the male is heterogametic for sex. The results were insufficient to permit any reliable estimate of the degree of impairment of the viability of the YY constitution.A method of producing single sex populations by the mating of sex reversed with normal fish is discussed.     

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.     

QTL for IHNV resistance and growth identified in a rainbow (Oncorhynchus mykiss) × Yellowstone cutthroat (Oncorhynchus clarki bouvieri) trout cross

Infectious hematopoietic necrosis virus (IHNV) is a major constraint to rainbow trout culture. Yellowstone cutthroat trout (Oncorhynchus clarki bouvieri) have greater resistance to this virus than do rainbow trout (O. mykiss), but the genetic mechanism of this resistance is not understood. We conducted a genome scan using a backcross of cutthroat trout into a rainbow trout background to estimate the number and locations of quantitative trait loci (QTL) associated with IHNV resistance and growth in trout. IHNV resistance was considered in terms of both survival (binary trait) and days to death (quantitative trait). The genetic map was scanned using interval mapping via two different approaches: one model considered survival alone and a second two-part model combined both survival and days to death. Three QTL were significantly (P ≤ 0.05) associated with virus resistance genome-wide, explaining 32.5% of the phenotypic variation. Cutthroat alleles at two of these QTL resulted in increased resistance to the pathogen, as expected. No growth QTL were detected in this cross. We suggest that these traits are genetically independent.     

Cytogenetic and histological studies of the brook trout, Salvelinus fontinalis (Mitchill), and the Arctic char, S. alpinus (L.) hybrids

Although brook trout and the Arctic char hybrids are able to reproduce, individuals with decreased fertility or even fish that are unable to produce any gametes have been also described. Abnormal gonadal development and disturbances in the gamete production in the char hybrid offspring may be triggered by the odd chromosome number and disturbances in their pairing during meiosis. To verify this hypothesis, cytogenetic examination and the gonadal histology analysis of the brook trout x Arctic char hybrids were carried out. Diploid chromosome number in the studied char (F₁) hybrids varied from 82 to 84 (FN = 99–102). Among 28 hybrids, 12 males, three females, nine intersex individuals and two sterile specimens were described. In the case of two individuals, gonads were not found. Diploid chromosome numbers in the males and intersex individuals varied from 82 to 84. Chromosome numbers in the females were 82 and 83 chromosomes. Two sterile fish exhibited karyotypes composed of 82 and 84 chromosomes. Predominance of the ovarian component in the intersex gonads and gonadal sex ratio distortion towards the males suggested hybrid females had problems with gonadal differentiation. However, the lack of the clear relationship between chromosome number and gonadal development in the studied hybrids did not support our hypothesis that odd chromosome number may be responsible for such reproductive disturbances in the hybrid individuals. We have presumed that sterility and intersexual development of the gonads may be caused by interactions between brook trout and Arctic char genes on the sex chromosomes and autosomes rather than unpairing of the parental chromosomes.     

Sex reversal in salmonid culture: Part III. The production and performance of all-female populations of brook trout

Oral administration of 17β-oestradiol at 20 mg/kg diet for a period from 0 to 60 days following “swim-up” resulted in the sex reversal of male brook trout and the production of essentially all-female populations. The gonads of these animals were indistinguishable from the ovaries of untreated fish.Oral administration of this hormone at the same concentration but for the period from 0 to 40 days resulted in a population which comprised 67% females, 21% males and 12% bisexual animals.Within the control group, the growth rate of males was significantly greater than that of females. Growth in the all-female stocks produced by dietary oestradiol administration (0–60 days) was significantly lower than that of the untreated population taken as a whole but was identical to that of the control females.The growth of brook trout during the first year was closely comparable to that of rainbow trout reared under identical conditions. During the second year, the growth of the rainbow trout exceeded that of the brook trout. At 19 months of age, all the animals in the control and oestradiol treated brook trout populations were fully mature; maturity in the rainbow trout was substantially confined to males.