Effect of triploidy on muscle cellularity and flesh quality of turbot (Scophthalmus maximus)

Production of triploid turbot is a relevant goal for the aquaculture industry due to the benefits associated with their sterility and growth. In order for a better understanding of the final quality of the species, muscle cellularity and quality parameters of the flesh were studied in seven diploid and seven triploid turbot after sexual maturation. White muscle cellularity was evaluated throughout white muscle cross‐sectional area from the epaxial left muscle section of each fish. To ascertain the flesh quality, several physico‐chemical parameters (moisture, ash, protein, total fat, fatty acids and minerals) were analysed, and textural mechanical properties (springiness, hardness, chewiness, cohesiveness, gumminess and adhesiveness) were determined objectively with a texturometer. Muscle cellularity was different between both groups, such that muscle fibre density was higher for diploid specimens (P < 0.001) and muscle fibres diameters were higher for triploids (P < 0.001). Both ploidy groups showed no significant differences in moisture, ash, protein and total fat content (P > 0.05). All textural properties analysed showed no significant differences between both ploidy groups (P > 0.05). In both ploidy groups, fibre density and fibre diameter showed no significant correlations (P > 0.05) with textural parameters such as springiness, cohesiveness, chewiness and adhesiveness. Ploidy did not affect fatty acid composition, with the exception of eicosapentaenoic acid (C_20:5ω3) which was higher in the triploid group. No significant differences (P > 0.05) existed between diploid and triploid turbot in content of all minerals analysed. According to our results, the ploidy status determines significant variations on muscle cellularity but does not influence the flesh quality of the turbot.     

Comparison of freshwater and marine performances of all‐female diploid and triploid Atlantic salmon (Salmo salar L.)

The performance of all‐female diploid (AF2N) and triploid (AF3N) Atlantic salmon were compared in fresh water, under commercial production conditions in 1995 and 1996 year classes. The performance of the 1996 year class was also assessed for 14 months in a commercial sea farm. Freshwater mortality was higher in the triploid groups. The majority of losses occurred in the early stages of egg development and during the first feeding period, when the incidence of non‐feeding fry was consistently higher. In growth studies, although diploid fry were significantly heavier during first feeding there were no significant differences in weight between groups some 8 months after fertilization or in presmolt growth periods from February to April in 1996 and 1997. Smolting rates were high (range 93.5–95.3%) and the incidence of deformities was low (< 1%) in both groups. Marine survival was lower in the triploid group, largely as a consequence of higher losses sustained during a period of chronic stress, when triploid losses were 9% higher. Growth patterns were similar for the first 11 months in sea water. Although graded triploid salmon were heavier in January 1998 (AF3N 1.62 ± 0.033 kg, AF2N 1.46 ± 0.36 kg, P < 0.05), when the fish were harvested in May 1998 diploid salmon were significantly heavier than triploid salmon although there was no significant difference in weights after evisceration (AF3N 2.40 kg ± 0.04 AF2N 2.49 kg ±0.03). The increase in weight of the diploids between winter and harvest reflects the growth spurt that occurs in maturing fish in the spring. Overall yields of triploid salmon in salt water were lower as a result of inferior survival.     

栉孔扇贝三倍体与二倍体的排泄研究

从温度变化、个体大小和昼夜变化等３个角度研究栉孔扇贝（Ｓｃｈｌａｍｙｓ ｆａｒｒｅｉ）三倍体与二倍体的排泄率。结果表明,栉孔扇贝三倍体与二倍体的排泄率均随温度升高而增大;不同壳宽排泄率（Ｎ）与温度（ｔ）的关系为：Ｎ＝７．２０３３ｅ＾０．１２１５ｔ,Ｒ＾２＝０．８４４１（三倍体ｓ组,壳宽约３．２ｃｍ）;Ｎ＝８．８１５３ｅ＾０．１０３１ｔ,Ｒ＾２＝０．９８６３（三倍体ｂ组,壳宽约４．３ｃｍ）;Ｎ＝６．     

Oxidative effect of l -carnitine on energy metabolism in diploid and triploid rainbow trout ( Oncorhynchus mykiss ): impact on metabolites

l-Carnitine plays a key role in the regulation of energy metabolism and growth in fish. The aim of this study was to determine the effects of dietary l-carnitine levels on carnitine homeostasis and energy metabolism in diploid and triploid trout (Oncorhynchus mykiss). Diploid and triploid trout (29.5 ± 0.6 and 31.8 ± 0.5 g, respectively) were fed with three diets supplemented with 15, 200 or 530 mg l-carnitine/kg, respectively, for 56 days. Compared to 15 mg l-carnitine, total carnitine content increased in the liver by 182% in diploid and by 154% in triploid trout fed 530 mg l-carnitine. In muscle, total carnitine content increased by 60 and 73% in diploid and by 34 and 75% in triploid with the increase in dietary l-carnitine levels. l-Carnitine caused no significant change in the plasma concentrations of metabolites like proteins (ammonia, urea and protein), lipid (triglycerides), carbohydrate (glucose, lactate) and enzyme activities (lipase, lactate, alanine transaminase, lactic acid dehydrogenase). Triploid trout had significantly lower plasma ammonia (P = 0.003), lipase (P = 0.005) and triiodothyronine (T3; P = 0.003) levels than diploid trout. In conclusion, ploidy significantly affected the energy metabolism in rainbow trout, dietary l-carnitine levels altered the l-carnitine homeostasis, but not influence nutritional metabolism.     

Changes in the pericellular connective tissue and breaking strength of the three types of muscles of the cultured carp Cyprinus carpio during storage in ice

To elucidate the structural changes in pink (P), white (W), and red (R) muscles during storage in ice, we measured the breaking strength and changes in pericellular connective tissues of cultured carp. The breaking strength just after killing was highest in R muscle (1.00 ± 0.20 N), lowest in W muscle (0.37 ± 0.07 N), and intermediate (0.84 ± 0.12 N) in P muscle. During the storage period, the breaking strength decreased first in R muscle, then in P muscle, followed by W muscle. The diameter of muscle fibers was greater in W muscle (113 ± 15 μm) than in P muscle (72 ± 3 μm) and R muscle (48 ± 2 μm). Destruction of the honeycomb structure of the pericellular connective tissue occurred most rapidly in W muscle and most slowly in R muscle. These results suggest that the interposing of P muscle fibers in the dorsal ordinary muscle contributes to the acceleration of post-mortem tenderization in fish.     

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.     

Effects of temperature on feed intake and plasma chemistry after exhaustive exercise in triploid brown trout (<Emphasis Type="Italic">Salmo trutta</Emphasis> L)

The physiological effect of temperature on feed intake and haematological parameters after exhaustive swimming in diploid and triploid brown trout (Salmo trutta) was investigated. Trout were exposed to an incremental temperature challenge (2 °C/day) from ambient (6 °C) to either 10 or 19 °C. Feed intake profiles did not differ between ploidy at 10 °C; however, triploids had a significantly higher total feed intake at 19 °C. After 24 days, each temperature–ploidy group was exposed to exhaustive swimming for 10 min. The haematological response differed between ploidy, with the magnitude of the response affected by temperature and ploidy. Post-exercise, acid–base and ionic differences were observed. Plasma lactate increased significantly from rest for both temperature and ploidy groups, but glucose increased significantly at higher temperature. Post-exercise, triploids at 19 °C had significantly higher osmolality and cholesterol than diploids, but differences were resumed within 4 h. Elevated alkaline phosphatase (ALP) and aspartate aminotransferase (AST) in fish at higher temperature suggested greater tissue damage; however, both ploidy responded similarly. Despite no significant differences in deformity prevalence, the type and location of deformities observed differed between ploidy (decreased intervertebral space with higher prevalence in tail area and fin regions for diploids, while vertebral compression, fusion in cranial and caudal trunks for triploids). These results suggest triploids have greater appetite than diploids at elevated temperature and that triploids suffer similar blood disturbances after exercise as diploids. These findings have implications for the management of freshwater ecosystems and suggest that stocking triploid brown trout may offer an alternative to diploid brown trout.     

The effect of ploidy on the fatty acid profile during the reproductive cycle of female rainbow trout (Oncorhynchus mykiss)

Rainbow trout Oncorhynchus mykiss triploids are regularly produced in fish farms to improve growth because the triploid females do not develop ovaries during the reproductive cycle. In this study, the tissue fatty acid allocations in triploid and diploid females were compared during the reproductive cycle to determine whether the ploidy influences the fatty acid profile of fish produced in aquaculture. The ovaries, liver, and white muscle fatty acid contents of diploid and triploid females were analyzed during the reproductive cycle. Diploid females tend to accumulate more polyunsaturated fatty acids than triploids during some phases of the reproductive cycle, and this profile was compensated by an increase in saturated and monounsaturated fatty acids in triploid females. Arachidonic acid (C20:4n6) was the main n6 polyunsaturated fatty acid in the ovaries of diploid females during the most advanced phases of the reproductive cycle, and docosahexaenoic acid (C22:6n3) was the main n3 polyunsaturated fatty acid. In triploid females, the percentage of both of these polyunsaturated fatty acids was lower than in diploid females during the most advanced phases of the reproductive cycle. In general, the lack of ovary development altered the hepatic synthesis of some fatty acids, mainly monounsaturated and polyunsaturated fatty acids, decreasing the content of the main fatty acids in the white muscle and, consequently, the mobilization of these specific fatty acids to the ovaries.     

Effects of safflower oil supplementation in diet on growth performance and body fatty acid composition of turbot (Psetta maxima)

The aim of the study was to investigate the effects of the diets that contain safflower oil and fish oil as lipid sources, on growth, feed conversion and body composition in turbot (Psetta maxima). Two iso-nitrogenous and iso-lipidic diets (55 % protein and 14 % lipid) were prepared that include 100 % fish oil (FO group) and 100 % safflower oil (SFO group) for turbots with average weight of 62.21 ± 1.28 g, and fish were fed with these diets for 104 days. At the end of the experiment, the weight gain, specific growth rate and feed conversion ratio were the highest in SFO group than in FO group (p < 0.05). Fatty acid composition of fish body reflected the fatty acid composition determined in the experimental diets. The amounts of palmitic acid (PA; C16:0), oleic acid (OA; C18:1n-9), linoleic acid (LA; C18:2n-6) and docosahexaenoic acid (DHA; C22:6n-3) were dominant fatty acids in fish body. It was confirmed that the usage of safflower oil instead of fish oil in turbot feed did not generate any negative effects on growth, feed conversion and the values regarding the growth performance.     

Morphology,sex steroid level and gene expression analysis in gonadal sex reversal of triploid female (XXX) rainbow trout (<Emphasis Type="Italic">Oncorhynchus mykiss</Emphasis>)

In non-mammalian vertebrates, estrogens and expressions of cyp19a1 and foxl2 play critical roles in maintaining ovary differentiation and development, while dmrt1 and sox9 are male-specific genes in testicular differentiation and are highly conserved. In order to deeply understand the morphological change, sex steroids level and molecular mechanism of triploid female gonadal reversal in rainbow trout, we studied the ovary morphology, tendency of estradiol-17β (E2) and testosterone (T) levels and the relative expressions of dmrt1, cyp19a1, sox9 and foxl2 in juvenile and adult fish. Our results demonstrated that the development of triploid female gonads in rainbow trout went through arrested development, oocytes dedifferentiation, ovary reconstruction and sex reversal finally. During early gonadal development (154–334 days post-fertilization), the expressions of foxl2 and cyp19a1 increased linearly, while expressions of dmrt1 and sox9 were extremely suppressed, and E2 level was higher, while T level was lower. During the mid-to-late period of triploid female gonadal development (574–964 days post-fertilization), the expressions of dmrt1 and sox9 remained high and were very close to the quantity of diploid male genes, and T levels were even reaching diploid male plasma concentrations, while expressions of cyp19a1 and foxl2 were decreased, leading to decrease in E2 level. We realized that the development model of rainbow trout triploid female gonads was extremely rare, and the regulatory mechanism was very special. Genes involved in gonadal development and endogenous estrogens are pivotal factors in fish natural sex reversal.     

Ethidium Bromide Nuclear Staining and Fluorescence Microscopy: An Alternative Method for Triploidy Detection in Fish

Abstract.– Erythrocyte nuclei from normal diploid (2N) and cold-shocked triploid (3N) Asian catfish Clarias macrocephalus were comparatively stained with fluorescent stain, ethidium bromide and with a bright-field stain, Giemsa. Stains were applied to dried blood smears on glass slides, with blood taken from fingerling fish without sacrificing the fish. Both stains produced comparable quality visual comparisons, with significant differences ( P < 0.001) between nuclear dimensions of 2N and 3N cells. Triploid nuclei were larger and more elongate than diploid nuclei. Karyotypic comparisons revealed that diploid fish had 54 chromosomes, while triploid fish had 81. Ethidium bromide nuclear staining required only 3 min after slides were air-dried and fixed, while Giemsa staining required more than 50 min.     

Tissue expression of PPAR-alpha isoforms in Scophthalmus maximus and transcriptional response of target genes in the heart after exposure to WY-14643

Peroxisome proliferator-activated receptors (PPARs) are involved in the regulation of lipid and carbohydrate metabolism and can be activated either by natural ligands as fatty acids or by synthetic ligands including several environmental chemicals. In this study, two PPARα isoforms (α1 and α2) were analyzed in turbot (Scophthalmus maximus) for a different tissue distribution. PPARα1 was ubiquitously expressed, while the PPARα2 was predominantly expressed in the heart. Following this result, turbot juveniles were exposed by injection to a synthetic selective PPARα agonist, WY-14643, for 14 days. Suppression subtractive hybridization (SSH) was performed with pools of heart samples of control and exposed fish to get insights into PPARα-regulated genes in the heart of juvenile turbot. Four genes were positively identified in the forward-subtracted and 12 genes in the reverse-subtracted cDNA SSH library, corresponding to the down-regulated and up-regulated genes in response to the WY-14643 treatment, respectively. The confirmation of these results in individual samples of juvenile turbot exposed to WY-14643 revealed a statistically significant mRNA induction of two cardiac muscle proteins (myosin light chain 2 and tropomyosin 4), which were shown to be involved in heart contraction and heartbeat regulation in other teleost species. Herewith, we showed for the first time that PPARα2 is predominantly expressed in the heart and that a PPARα agonist can induce the mRNA expression of cardiac muscle proteins in teleosts.     

High dietary lipids elevate carbon loss without sparing protein in adequate protein-fed juvenile turbot (Psetta maxima)

This study was undertaken to determine the effects of dietary lipid levels on growth and nutrient utilization, and carbon (C), nitrogen (N) and phosphorus (P) balances in juvenile turbot (Psetta maxima) in brackish water. Four isonitrogenous diets (50 % protein) differing in dietary lipid levels from 10 to 19 by 3 % increments (named 10L, 13L, 16L and 19L, respectively) were fed to four replicate groups of fish with an initial weight of 54.4 ± 0.2 g for 9 weeks. Significantly better growth and feed conversion ratio were observed in fish fed 13L than those fed 16L and 19L. The whole body, fillet, viscera and liver dry matter and lipid levels were increased as dietary lipid was increased. Liver protein levels were lower in 16L and 19L than 10L. There was an ascending pattern in the whole-body C levels and C/N ratios with the increase in dietary lipids. No significant effect of dietary lipids on either protein efficiency ratio or N and P retentions was detected. Dietary lipids led to an increase in C losses to the environment. Overall, high dietary lipids when introduced at an adequate protein level did reduce growth and feed utilization without a protein-sparing effect and resulted in higher C losses in juvenile turbot reared, and a dietary lipid level of about 13 % seemed to be optimum.     

不同倍性长牡蛎闭壳肌和精子的组织学和超微结构观察

以二倍体、三倍体、四倍体长牡蛎(Crassostrea gigas)闭壳肌和精子为研究对象, 采用石蜡组织切片、透射电镜和扫描电镜方法, 比较了 3 种倍性长牡蛎闭壳肌和精子的组织学特性及超微结构的差异。研究结果显示, 二、三、 四倍体闭壳肌组织的肌纤维都包含粗肌丝、细肌丝、肌膜、肌束膜、肌内膜、线粒体、囊泡、小管、细胞核、肌浆网等结构, 但三者的形态及超微结构不同, 其中三倍体闭壳肌线粒体直径、肌纤维直径、粗肌丝密度显著大于二倍体和四倍体, 肌纤维密度显著小于二倍体和四倍体。不同倍性精子的扫描电镜和透射电镜结果显示, 二、三、四倍体长牡蛎精子在组成结构上相似, 分别由头部、中段和尾部构成, 但不同倍性精子中各结构大小和线粒体数目不同。在长牡蛎精子头部长度、宽度, 顶体高度、宽度, 线粒体长度的测量分析中, 四倍体精子的头部、顶体以及线粒体显著大于二、三倍体。三倍体精子的头部和顶体显著大于二倍体, 但三倍体精子的线粒体大小与二倍体相比无显著差异。此外, 二、三倍体精子均含 4 个线粒体, 但在本研究中发现了二倍体精子含有 5 个(占比 1%)线粒体的特例。四倍体长牡蛎精子的线粒体数目不同, 含有 4 个(占比 46%)、5 个(占比 52%)、6 个(占比 2%)这 3 种情况。 该研究阐明了不同倍性长牡蛎闭壳肌及精子的组织学特性和超微结构, 为深入研究长牡蛎肌纤维生物学特性和精子繁育生物学提供了基础资料。     

Optimal conditions for cold-shock induction of triploidy in red tilapia

Production of sterile triploid red tilapia [Oreochromis mossambicus (Mozambique tilapia); Peters, 1852 × Oreochromis niloticus (Nile tilapia); Linnaeus, 1758] is an effective strategy to overcome their prolific breeding. Optimal conditions for cold-shock induction of triploidy in red tilapia were investigated by experimentally examining two variables: appropriate temperature of the shock and duration of shock treatment. A constant time after insemination of 4 min was used to determine the best combination of temperature (6, 7, 8, 9, 11, 13, 15 °C) with different durations of shock (10, 20, 30, 40, 50 min) with resultant ploidy level verified karyotypically. Shock duration for 30 min at a temperature of 9 °C was found most effective in producing maximum triploidy (98.7 %) with higher rates of hatching (63.2 %) and survival up to yolk-sac stage (75.8 %). The chromosome count confirmed that triploid percentages were higher when cold shock was used for longer durations at each temperature; however, hatching rates were generally decreased. The maximum triploid yield (82.1 %) obtained was higher than the yield obtained using heat shock (72.7 %) in red tilapia previously. The application of the results of this study has the potential to greatly improve the production of triploid red tilapia in commercial aquaculture.     

Effect of Stock Density and Ploidy in Jundia,Rhamdia quelen,Larvae Performance

The performance (growth and survival) of diploid and triploid jundia, Rhamdia quelen, was evaluated at six different stocking densities (10, 60, 110, 160, 210, 260 larvae/liter) during 31 days after rearing in an intensive larviculture system. Triploid fish exhibited a significantly higher survival rate than diploids at all stocking densities (27.1 ± 4.3% vs. 12.1 ± 3.3%; P < 0.01). Survival was not affected by stocking density (P > 0.05). Length gain was not affected by either ploidy or stocking density. Diploid fish gained more weight than triploids (P < 0.05), though this difference could result from lower fish densities in diploid treatments resulting from the higher mortality rate of diploid fish. This hypothesis is strengthened by the higher biomass present in triploid treatments (P < 0.01).     

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.     

Differences between Atlantic halibut (Hippoglossus hippoglossus L.) and turbot (Scophthalmus maximus L.) in tolerance to acute low temperature exposure

Juvenile Atlantic halibut (Hippoglossus hippoglossus L.) and turbot (Scophthalmus maximus L.) acclimated to 8–9 °C sea water were transferred to sea water at 1 °C for 7 days. An immediate spasm-like response was noticed in the turbot after the transfer, but not in the halibut. None of the fish died. The turbot suffered from substantial physiological disturbances after the transfer. Their blood plasma Cl^– concentration increased and muscle water content decreased markedly. The plasma glucose concentration was several times higher than the control level from day one onwards. A decrease in the haematocrit was recorded some hours after transfer, followed by a marked increase on day 7. In halibut, the plasma Cl^– concentration fell somewhat during the first days, but returned thereafter to its pretransfer level. The muscle water content was unchanged. Both haematocrit and plasma glucose concentration were unchanged until day 7, when both were significantly higher. The differences in the response to low-temperature challenge between halibut and turbot are probably related to genetic differences in temperature tolerance, which reflect differences in the distribution of the species.     

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.