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.     

Reproductive performance and mature gonad morphology of triploid and diploid Black Tiger shrimp (Penaeus monodon) siblings

Sibling harvest age Black Tiger shrimp triploids and diploids of both sexes were reared to reproductive maturity, crossed with wild caught females and males, conditioned for spawning and a comprehensive reproductive performance trial was undertaken. Ovarian development, spawning frequency, fecundity, hatch rate, gonad morphology, male reproductive tracts and thelycum impregnation rates of the wild female × triploid male cross were assessed. After ablation, ovarian development and cycling between wild G₀ diploid and G₁ diploids was not significantly different, whereas G₁ triploids failed to show any signs of ovarian development and cycling, thus resulting in no G₁ triploid female spawnings. There were 10 G₀ diploid female × G₀ diploid male first‐spawnings and 9 G₀ diploid female × G₁ diploid male first‐spawnings, all of which produced viable nauplii. In comparison, there were 7 G₀ diploid female × G₁ triploid male first‐spawnings, none of which produced viable nauplii. The 26 wild G₀ diploid female spawnings had more eggs than the 1 G₁ diploid female spawning. Gonad morphology and male reproductive tract assessments showed impaired reproductive development in triploid gonadal tissues of both sexes (compared with sibling diploids and wild shrimp) to a point where complete maturation had not occurred. The thelycum of 16 wild G₀ diploid females crossed with G₁ triploid males had no visible spermatophore present, suggesting that G₁ triploid males are incapable of developing viable spermatophores and mating with females. This study demonstrates that the triploid females and males are incapable of producing viable gametes and are thus reproductively sterile.     

Hatchery production of diploid and triploid clams, Tapes dorsatus (Lamarck 1818): a potential new species for aquaculture

To assess the potential for hatchery production of the venerid clam, Tapes dorsatus, diploid and triploid clams were produced and ongrown until the spat reached 5–8 mm in size. Triploidy percentages at metamorphosis that ranged from 56 to 85% were induced using a 15-min exposure to 1 mg/l cytochalasin B. No differences were observed in the growth rate of sibling diploid and triploid T. dorsatus larvae. Survival of diploid clams to pediveliger stage was however higher. Post-metamorphic growth and survival of diploid and triploid clams remained the same until the clams were removed from the hatchery to outdoor nurseries. Throughout the larval and early spat phases of production, T. dorsatus growth was similar to that reported for the Manila clam, Tapes philippinarum. Based upon its ease of culture, rapid growth and marketability, T. dorsatus are thought to have considerable aquaculture potential.     

Farming triploid oysters

Although the commercial benefits of triploidy have been evaluated in the Pacific oyster Crassostrea gigas (Thunberg, 1793), eastern oyster C. virginica (Gmelin, 1791), Sydney rock oyster Saccostrea glomerata (Gould, 1850) and European flat oyster Ostrea edulis (Linnaeus, 1750), so far this technique has only been commercialised for Pacific oysters.

Commercial production of triploids on the West Coast of North America began in 1985. Since then production of triploids has greatly increased and the use of tetraploid males to fertilise eggs from diploids to produce batches of 100% triploids has been developed. In 1999/2000, triploid Pacific oysters made up 30% of all Pacific oysters farmed on the West Coast of North America. Some hatcheries now use tetraploid males instead of chemical or physical stress to produce triploids. The rapid uptake of triploid and tetraploidy techniques has been facilitated by the almost total dependence that these oyster industries have on hatcheries for the supply of seed. This industry in the Pacific Northwest of the US and in British Columbia, Canada, would not have developed to its current size without hatchery seed supplies. Triploids are preferred over diploids in summer because diploids are less marketable when in spawning condition.

There was only limited interest in triploidy production in France until 1999, when IFREMER began to make sperm from tetraploids available to commercial hatcheries. In 1999/2000, only 10% to 20% of all the hatchery-supplied Pacific oyster spat were triploids, but with the use of sperm from tetraploid oysters, this could increase sharply.

Elsewhere around the world, the commercial uptake of triploid oysters has been slow to develop. However, in countries where the production of Pacific oysters is based on hatchery supply of seed, it is likely that with the use of tetraploid oysters, the farming of triploid oysters will increase in the near future.     

Growth and gonadal development in diploid and triploid Atlantic cod (Gadus morhua)

Induction of triploidy has been suggested as an effective tool to prevent spawning of farmed fish. This experiment examined the growth potential of triploid cod when reared communally with diploid ones after the juvenile stage. Pressure treatment was used to induce triploidy in a batch of cod eggs in April 2009. The resulting offspring were reared separately from their diploid counterparts until they reached the proper size for PIT tagging. At the age of 8 months, an equal number of 115 diploids (135.5 ± 3.95 g) and triploids (93.6 ± 2.63 g) were communally reared in a circular flow-through tank until the age of 22 months. By the end of this rearing period, diploids (1,002.4 ± 39.9 g) were significantly heavier than triploids (654.6 ± 27.7 g), but the specific growth rate did not differ significantly during the growth trial. Gonadal development at the age of 22 months was also lower among triploids than diploids, especially for females (5.3 and 91.9 %) but also for males (32.5 and 72.7 %). Sterility among female triploids was evident by the reduced size and dysfunctional gonads, but gonadal development in male triploids was less suppressed. Prevalence of body deformities was, however, significantly higher among triploids (62.6 %) than diploids (33.9 %). Higher prevalence of deformities in triploid cod underlines the need for further fine-tuning of the triploidization procedure or finding other methods of sterilization. At present, triploid cod are still far from being established as an alternative for commercial production.     

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.     

Growth and gonadal development in diploid and triploid turbot (Scophthalmus maximus)

This study determined the effect of triploidy on the survival, growth and gonadal development of turbot from 6 to 48 months of age. From 6 to 24 months of age (first sexual maturity), survival was similar in both ploidies (P > 0.05). From 24 to 48 months of age, after the first sexual maturity, survival was 91.9% in diploids and 100% in triploids, which did not exhibit the post-spawning-associated mortality. Growth was similar for both ploidies during the first year of life. After that, triploids grew significantly (P < 0.05) more that diploids, with more marked differences after each spawning season. From 24 to 48 months, the average weight difference between both ploidies was 11.4 ± 1.9%, ranging from 4.3 to 23.0%. At 47 months of age, the biomass of triploids was 10.3% greater in total weight and 14.3% greater in eviscerated weight. Gonads of triploid males were similar to that of diploids, whereas in triploid females, they were significantly smaller and rudimentary. A histological analysis carried out at 47 months of age showed complete sterility of triploids in both sexes. Sex ratio was 1 male (M):0.6 female (F), for diploids, significantly (P < 0.05) different from 1:1, and 1 M:3.3 F for triploids, significantly (P < 0.05) different from 1:1 and from the diploids. Since females grow more than males, culture of triploids benefited from the high female ratio, which helped to reduce size dispersion. In addition, their sterility allowed better performance by avoiding the reduction in growth that takes place during the spawning periods. Together, these observations indicate that triploidy induction can be an interesting option for turbot aquaculture, especially for the production of large-size fish of more than 2 years of age.     

Production of triploid eastern little tuna,Euthynnus affinis (Cantor, 1849)

Herein, we developed a triploidization method using spawned eggs collected immediately after spawning of eastern little tuna (ELT), Euthynnus affinis. ELT broodstock induced the spawning by hormonal treatment in the tank, with the resulting spawned eggs being used for the triploidy induction. Under optimal conditions, the mean ± SEM triploidization and hatching rates were 97.2 ± 2.8% and 84.5 ± 10.3%, respectively. Although triploid ELT showed growth performance equivalent to that of diploids, the triploids died at a higher rate than the diploids during 2–4 weeks post‐hatching when triploids and diploids were reared in the same tank. Therefore, we propose that it would be necessary in a practical operation to use triploid‐only ELT seedlings to avoid selective cannibalism by the diploids. The ELT triploids exhibited an all‐female phenotype. Because previous studies have reported that female triploids show a greater probability of sterility than male triploids, this characteristic could be a major advantage. Since this triploidization method, using spawned eggs, can be performed without handling the broodstock, it is possible to avoid the physical damage caused by the process of artificial insemination, making it possible to repeatedly produce triploid populations without valuable broodstock loss. Thus, we have developed an efficient method to produce ELT triploids, although further study is essential to evaluate sterility of the triploid ELT.     

Preliminary study on performance of triploid Thai silver barb, Puntius gonionotus

The triploid chromosome condition was induced in Thai silver barb (Puntius gonionotus) by application of cold shock (2°C) to eggs at time intervals after activation of 0.5 min with a duration of 10 min which resulted in mean triploidy yield of 72.5% at 9 months of age. Growth rate of the 2–9-month-old, cold shock group (0.1–6.2 g/month) did not differ from that of the control (0.1–5.7 g/month). Gonadal somatic indices (GSI) of presumed triploid males and females were lower than that of control (GSI values of the presumed triploids were 35.0–60.2% and 28.7–75.9% of control males and females, respectively). Spermatogenesis and oogenesis were retarded in triploids. However, all stages of spermatogenic cells were observed in triploid males, including few spermatozoa. Oocytes of triploid females did not undergo vitellogenesis while normal oogenesis was observed in diploids. Nuclear volume of red blood cells (RBCs) of triploid fish was 1.63 times larger than that of diploids.     

Hormonal profile of growing male and female diploids and triploids of the blue tilapia,Oreochromis aureus,reared in intensive culture

Triploidy as a result of thermal shock exposure of fertilized eggs decreases the growth rate ofOreochromis aureus as compared to their diploid controls, but this is due to the higher female ratio present in triploids (86%) and the lower growth rate of females. When females and males are considered separately, the growth rate is not significantly different in diploids and triploids. Since triploidy results in a malfunctioning steroidogenesis in females (mainly testosterone (T) and 17β-estradiol (E₂)), but does not affect the growth rate, it is concluded that female gonadal steroids do not influence growth unless in pharmacological concentrations. These low levels of gonadal steroids are generally accompanied by higher levels of gonadotropin (GtH), but the difference is not always significant. Despite their lower growth rate diploid females have higher plasma concentrations of growth hormone (GH) during several months compared to the triploid females and diploid males. 3,5,3′-triiodo-L-thyronine (T₃) levels, however, are comparable between diploid and triploid females (except for 1 month), but higher in diploid males in 4 of the 5 months studied. 11-ketotestosterone (11kT) is always higher in males. These results indicate that the higher growth rate of males may be related to the high circulating levels of T₃ and 11kT.     

Comparative Growth of Diploid and Triploid Asian Catfish Clarias macrocephalus in Thailand

Wild caught Asian catfish were spawned manually following HCG injection, and a portion of the eggs were subjected to cold-shock at 4 C for 15 min within 2-min post-fertilization. Nuclear diameter measurements of cold-shocked fish revealed that 96% were triploids (3N), while non-shocked fish were all diploids (2N). During larval and fry culture (first 26 d), triploid fish mortality was =50%, while diploid mortality was =25%. Following 8-mo culture in tanks at three stocking densities, triploid fish survival was significantly greater ( P < 0.05), than diploids, with 84.0% and 57.3%, respectively. Triploid live weight was also significantly greater than diploids, with 69.2 and 45.9 g averages, respectively. Ninety-two percent of diploids had welldeveloped gonads after 8 mo; whereas none of the triploids had mature gonads. Gonads were undifferentiated with 31% of the triploids. These sexually undifferentiated fish had greater growth rates than male or female triploids, and greater growth than all diploids. Carcass weight (gutted) of triploids was 95.8% of live weight, compared with 92.5% for diploids. Lastly, triploids had very few deformities compared with diploids, with 1.3% and 17.6%, respectively. Deformities included curved spines, and humped backs just posterior of the head.     

Production of all-female triploid rainbow trout

Triploid rainbow trout were produced by heat shock treatment of eggs soon after fertilization with either normal sperm or sperm from masculinised females. The proportion of triploid fry, as judged from red blood cell nuclear volume, varied between 75% and 100% in three experiments using different batches of eggs from an autumn-spawning strain of trout while a single batch of eggs from a winter-spawning strain yielded 50% triploids.

A microscopic examination of the gonads was made on 5-month-old fish weighing between 1 and 3 g. In female controls the ovaries were packed with oocytes while those from female triploids, although showing the typical lamellar structure of an ovary, contained no oocytes, thus indicating that female triploids are sterile. The testes from triploid males appeared to be developing normally.

The use of masculinized females combined with heat shock treatment of eggs to produce triploids, allowed the production of sterile all-female triploids. This should have considerable potential for aquaculture.     

Added value from an added chromosome: Potential of producing large fillet fish from autumn to spring with triploid rainbow trout,Oncorhynchus mykiss

Three‐summers‐old all‐female triploid and diploid rainbow trout were compared after one on‐growing season in sea net cages. Slaughter traits of round weight, gutted weight, fillet weight, carcass% and fillet% were measured at three times in November 2017, January and April 2018. The triploid group had lower daily growth coefficient mean (4.25) and higher feed conversion ratio (1.18) than diploids (4.48 and 1.05, respectively) during on‐growing (June–November). In November, no difference of means was found between mature or immature diploids and triploids for any of the weight traits when the effect of vertebrae defects was statistically removed. However, the triploids had attained higher means than mature or immature diploids in gutted and fillet weight by January, suggesting that the loss of muscle mass during early winter was lower in triploids. Sexually maturing diploids (46%) had lower slaughter yield means compared to triploids or immature diploids at each measurement time, and these differences also increased during overwintering. Instead, the means of yield traits remained similar between the triploid and immature diploid groups through the winter. Likewise, fillet redness remained at equally high level in triploids and immature diploids, whereas in maturing diploids this attribute decreased substantially during overwintering. The triploid group had a higher incidence of vertebral defects (12.0%) than diploids (5.3%). The present results demonstrate the potential of triploid trout in producing large‐sized (>2 kg) fillet fish until spring markets. However, more detailed investigations are needed, particularly regarding the animal health and growth efficiency in triploids, relative to their diploid counterparts.     

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.     

The reproductive physiology of three age classes of adult female diploid and triploid brook trout (Salvelinus fontinalis)

Triploid female fish show impaired gametogenesis and are unable to produce viable offspring. The reproductive physiology of artificially-induced triploid female salmonids has been well described up until the time of first sexual maturation in diploids, but few reports exist for older triploids. This study reports the influence of triploidy on growth, ovarian development and reproductive endocrinology among three age classes of female brook trout (Salvelinus fontinalis) in comparison to sibling diploids. Triploids were larger than diploids for most of the study period, but the difference was statistically significant only during maturation and spawning of 2⁺ diploids. Plasma estradiol-17 (E₂), testosterone (T) and vitellogenin (VTG) levels in triploids were generally lower than in diploids, and VTG was the only parameter to show seasonal fluctuations resembling those of diploids. Triploids showed significantly lower GSI and total oocyte number than diploids of similar age, and only half of all triploids sacrificed during the study (n=56) had developing oocytes in their ovaries. At age 3⁺, 13 of 19 triploid females had oocytes at various stages of development, including perinucleolar, yolk vesicle and yolk globule stages. In addition, three of these fish had collectively produced 72 mature stage oocytes. Thus, whereas diploid brook trout can produce mature oocytes as two-year-olds, triploids cannot do so until four years of age, with the number of mature oocytes being greatly reduced.     

The growth and reproductive endocrinology of adult triploid Pacific salmonids

This paper describes the effect of triploidy on growth and reproductive endocrinology in the months leading up to and including spawning in rainbow trout,Salmo gairdneri, and pink salmon,Oncorhynchus gorbuscha. Growth rates were the same for diploid and triploid rainbow trout, but triploid female pink salmon were smaller than maturing diploid females and diploid and triploid males of the same age. Triploid males of both species developed typical secondary sexual characteristics and had normal endocrine profiles, although their cycle appeared to be delayed by about one month. Triploid females remained silvery in appearance and showed no endocrine signs of maturation, even at the level of the pituitary. Thus, although triploids of both sexes are genetically sterile, only the females do not undergo physiological maturation.Reported in part at the Third International Symposium on Reproductive Physiology of Fish, St. John's, Newfoundland, August 2–7, 1987.     

Hemoglobin level,metabolic rate,opercular abduction rate and swimming efficiency in female triploid brook trout (Salvelinus fontinalis)

Metabolic (oxygen consumption) rate, opercular abduction rate and tail beat frequency were determined in two strains of diploid and triploid female brook trout (Salvelinus fontinalis) while these fish swam at 0.37±0.02 body lengths per sec in a Blazka respirometer. Total blood hemoglobin level was also measured and opercular condition examined. Total blood hemoglobin levels in diploids and triploids were equal. The opercular abduction rate was the same in diploids and triploids (regardless of whether triploid opercular condition was good or poor) yet triploids had a lower oxygen consumption rate than diploids, indicating that triploids take up less oxygen than diploids per opercular cycle. Tail beat frequency, an indicator of swimming effort, was the same in diploids and triploids, suggesting that triploids require less oxygen than diploids for a similar swimming effort.     

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.     

Triploid hard clams Mercenaria mercenaria produced by inhibiting polar body I or polar body II

Two types of triploid hard clams Mercenaria mercenaria were produced by inhibiting polar body I (PB1) or polar body II (PB2) with cytochalasin B. Treatments were applied at 22–23°C, with PB1 inhibition starting at 4–7 min postfertilization and ending when PB2 was first observed in control groups, and with PB2 inhibition starting at 17–23 min postfertilization and ending when 80% of control eggs released PB2. Triploid induction success was evaluated by chromosome counting in 2–4 cell embryos and by flow cytometry at larval and juvenile stages. PB2 inhibition produced more triploids (82%–100%) than PB1 inhibition (71%–83%), although the difference was not significant (p ≥ .088). Triploid percentages in PB1‐ or PB2‐inhibited groups showed a small but insignificant decline during the first 6 months. At month 3, PB1 and PB2 triploids were not different from their within‐group diploids, but significantly larger than control diploids; PB1 triploids were significantly larger than PB2 triploids (p ≤ .003). At month 6, PB1 triploids were not different from either within‐group or control‐group diploids, while PB2 triploids were significant larger than both within‐group and control diploid; PB1 triploids were smaller than PB2 triploids. At month 16, PB1 and PB2 triploids in one remaining replicate were not different from their within‐group diploids. Overall, this study shows that triploids can be efficiently produced by PB1 or PB2 inhibition, and their growth performance relative to diploids is variable depending on age and replicates or parental genotype.     

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.