Skeletal anomaly assessment in diploid and triploid juvenile Atlantic salmon (Salmo salar L.) and the effect of temperature in freshwater

Triploid Atlantic salmon tend to develop a higher prevalence of skeletal anomalies. This tendency may be exacerbated by an inadequate rearing temperature. Early juvenile all‐female diploid and triploid Atlantic salmon were screened for skeletal anomalies in consecutive experiments to include two size ranges: the first tested the effect of ploidy (0.2–8 g) and the second the effect of ploidy, temperature (14 °C and 18 °C) and their interaction (8–60 g). The first experiment showed that ploidy had no effect on skeletal anomaly prevalence. A high prevalence of opercular shortening was observed (average prevalence in both ploidies 85.8%) and short lower jaws were common (highest prevalence observed 11.3%). In the second experiment, ploidy, but not temperature, affected the prevalence of short lower jaw (diploids > triploids) and lower jaw deformity (triploids > diploids, highest prevalence observed 11.1% triploids and 2.7% diploids) with a trend indicating a possible developmental link between the two jaw anomalies in triploids. A radiological assessment (n = 240 individuals) showed that at both temperatures triploids had a significantly (P < 0.05) lower number of vertebrae and higher prevalence of deformed individuals. These findings (second experiment) suggest ploidy was more influential than temperature in this study.     

Studies on triploid oysters in Australia: farming potential of all-triploid Pacific oysters, Crassostrea gigas (Thunberg), in Port Stephens, New South Wales, Australia

Triploid Pacific oysters Crassostrea gigas farmed in Port Stephens, NSW had an exceptionally fast growth rate and reached a whole weight of 55 g in 13 months versus 20 months for diploids. Mortality of the triploids (24.5±2.94%) was significantly lower (P<0.05) than that of the diploids (40.0±2.26%) over the duration of the experiment (July 2002–February 2004). Unfortunately, this advantage was offset by discoloration of the meats of the triploids when they were in better condition than the diploids over summer (October 2003–March 2004). However, discoloration of meat of triploids had cleared up by April 2004 and neither did they suffer this problem from April–September 2003. The triploids also had a lower peak condition than the diploids. Oysters in peak meat condition, i.e. spawning condition, are preferred for the half shell trade in Australia and in this study, there was at least a six‐month period prior to discoloration, when the triploids were large enough and had sufficient meat condition for marketing on the half shell.     

Comparative performance of growth,vertebral structure and muscle composition in diploid and triploid Paralichthys olivaceus

Growth, skeletal structure and muscle composition of cold‐shock‐induced triploid olive flounder Paralichthys olivaceus were investigated. The average values of total length and total weight of triploids were higher than those of diploids from 5 to 11 months posthatch (mph). The growth difference disappeared after 11 mph. The skeletal structure of flounder at 11 mph was observed by X‐ray imaging method. There are four kinds of vertebral deformity including vertebrae fusion, one‐sided compression, two‐sided compression and vertically shifted. The trunk region (V8–18) and tailing end of the vertebral column were the predominant locations of deformity. In general, the frequencies of vertebral deformities in triploids (60.0%) were higher than those in diploids (33.3%, p < 0.05). Both the number of fish with deformed vertebrae and the average frequencies of deformed vertebrae in triploids were significantly higher than those in diploids (p < 0.05). The muscle tissues of diploid and triploid flounder at 11 mph contain the same types of fatty acid and free amino acid profiles. The number of fatty acids with significant higher contents in diploids and triploids was one and ten, respectively (p < 0.05). The contents of free amino acids showed no difference between triploid and diploid fish.     

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.     

Effect of ploidy on the mortality of Crassostrea gigas spat caused by OsHV‐1 in France using unselected and selected OsHV‐1 resistant oysters

The effect of ploidy on the mortality of Crassostrea gigas spat caused by the ostreid herpesvirus (OsHV‐1) genotype μVar was investigated at five sites along the Atlantic coast in France in 2011. Sibling diploids and triploids were produced using either unselected or selected OsHV‐1‐resistant oysters. No significant interactions were found between the factors of environment, genotype and ploidy at the endpoint dates. The mean mortality rates at the sites were 62% and 59% for diploids and triploids, respectively, and the two rates were not significantly different. The mean mortality rates were 33% and 32% for sibling diploids and triploids, respectively, when OsHV‐1‐resistant parents were used and 91% and 85%, respectively, when unselected parents were used. The results were confirmed through other broodstocks tested in 2013. Our study is the first to clearly show that mortality related to OsHV‐1 is similar between diploids and triploids in C. gigas when the same germplasm is used for both ploidy. Furthermore, OsHV‐1 resistance was not substantially altered by triploidization, indicating that the achieved selective breeding of diploid oysters for OsHV‐1 resistance can be translated into improved survival in triploids.     

Blocking polar body with cytochalasin B in the fertilized eggs of the small abalone, Haliotis diversicolor supertexta (Lischke), and the development and ploidy of the resultant embryos

The effects of blocking polar body I (PB1) or polar body II (PB2) with four different dosages of cytochalasin B (CB) on the development and ploidy of resultant embryos were studied in the small abalone, Halitis diversicolor supertexta (Lischke). To block the release of PBI, the fertilized eggs were treated with 0.25, 0.5, 1.0 or 2.0 mgL^?1 of CB for 10min beginning at 3 min post-fertilization at 24°C. To block the release of PB2, the fertilized eggs were treated under the same conditions as PB1, except that the treatment was begun 10min post-fertilization. In the control group, only 41.8% of the cells had a diploid number of 32 chromosomes, although spontaneous haploids (9.0%). tripolids (7.5%) and aneuploids (41.7%) were also observed. In CB treatment of PB1 and PB2 groups. 5.0-28.6% of the cells remained as diploid. triploids (10.0-18.9%) and aneuploids (41-1-61.0%). With regard to the development of the resultant embryos, the proportion of normal embryos in the control group was 87%, while in the treatment groups, the proportions of normal embryos in the FBI and PB2 groups were 57-58% and 53-56% in the 0.25 mg L^?1 and 0.5mg L^?1 CB treatments, respectively. From this data on induced triploids and the resultant development of normal embryos, the proportions suggest that 0.25-0.5 mg L^?1 of CB for 10min was sufficient for blocking the release of FB1 or PB2 to produce triploids in the small abalone.     

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.     

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.     

Plasma levels of Gonadotropin-II and gonadal sex steroids in triploid catfish, Heteropneustes fossilis (Bloch)

Triploid fish have under-developed gonads due to altered reproductive endocrinology. Triploids of Indian catfish (H. fossilis) showed significantly reduced plasma levels of gonadotropin (GtH-II), testosterone (T) and estradiol-17 (E₂) than that of diploids throughout the year, except for the resting phase, irrespective of sex. Plasma levels of GtH-II were significantly different (p<0.001) between diploid and triploid fish during preparatory, prespawning and spawning phase. The plasma testosterone contents in triploids were significantly less (p<0.001) than that of diploids, except during the resting phase. Triploid females showed very low titres of estradiol-17 (<1 ng ml^–1) throughout the annual reproductive cycle in contrast to highly fluctuating levels in diploid females. Thus, this study for the first time provides information on reduced levels of GtH-II and sex steroids in plasma of male triploid fish and additional information on species-specific alteration of sex hormones in female triploids.     

A comparative study of growth and feed utilization efficiency of sex-reversed diploid and triploid Nile tilapia, Oreochromis niloticus L.

Growth performance, survival and feed utilization of diploid (2n) and triploid (3n) sex‐reversed male and female Nile tilapia were evaluated at maintenance feeding (1% body weight (BW) day^?1), fixed feeding (3% BW day^?1) and apparent satiation feeding levels in a freshwater recirculation system comprised of thirty‐six 1‐m³ concrete tanks at the Asian Institute of Technology, Bangkok, Thailand. Triploid Nile tilapia (3n) was produced by subjecting fertilized diploid (2n) tilapia eggs to heat shock. After hatching, fish were sex‐reversed to all‐male and all‐female populations by oral administration of 17 α‐methyltestosterone (60 mg kg^?1 feed) and ethynylestradiol (100 mg kg^?1 feed) respectively. There was significantly higher growth with increased ration levels in both male and female groups. There were no significant differences in final BW, specific growth rate, survival rate, feed conversion ratio and protein efficiency ratio between diploid and triploid fish. Triploids had lower gonad weights than diploids, and this was particularly evident at the satiation feeding level. Triploid fish had a significantly higher apparent net protein utilization and percentage of gutted weight than diploids at all feeding levels. Higher protein utilization efficiency of triploids might be an advantage for commercial tilapia culture but further research is necessary to make such a conclusion.     

Muscle growth of triploid Atlantic cod juveniles (Gadus morhua)

This study has investigated the muscle growth of diploid and triploid Atlantic cod (Gadus morhua) juveniles raised in replicate tanks over a period of 29 weeks and analysed at three sampling points (February, June and September). Data for weight, length, condition factor (K), muscle fibre growth and myogenic progenitor cells (MPCs) number were collected and results were analysed in relation to body growth and ploidy status. Diploids were significantly heavier than triploids throughout the trial (~10–20%) and had K in June and September samplings. Over the whole period, the rate of muscle fibres' recruitment was 318 fibres per day and 252 fibres per day for diploid and triploid cod respectively. The larger body weight of diploids resulted in a total number of fast fibre number of 114 979 compared to 91 086 in triploids. The average diameter of the 2.5% of the smallest fibres (2.5th percentile) was higher in diploids than triploids at the start of the trial, with a reversed picture for the average of the upper 2.5% (97.5th percentile) at the end of the trial. The probability density function of the estimated muscle fibre diameters showed similar fibre size distribution between size‐matched diploids and triploids at all sample points. The peak fibre diameter was approximately 25 μm in February and increased to approximately 50 μm in June and September, irrespectively of ploidy. Pax 7 were used as molecular markers for MPCs. A positive correlation between Pax 7⁺ cells and total body length was observed only among triploid fish at the onset of the experiment.     

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.     

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.     

Larval metamorphosis and morphological characteristic analysis of triploid shrimp Fenneropenaeus chinensis (Osbeck, 1765)

Both MI and MII triploids were successfully produced by heat shock in Chinese shrimp Fenneropenaeus chinensis. The inducing conditions for MI and MII triploids were optimized. The highest inducing rate obtained for MI triploids reached more than 90%, and that for MII triploids reached nearly 100% at the nauplius stage as evaluated using flow cytometry. Comparisons of survival rates at larval stages between triploids and diploids or diploids experiencing treatment and diploids without treatment were performed. At larval stage from nauplii to postlarvae, heat shocks lowered survival at larval stages even if the ploidy was not changed. Ploidy did not affect shrimp larvae survival, and no significant difference was found in the survival of shrimp larvae between MI and MII triploids. Highly significant differences were observed in the morphology of triploids and diploids, and no apparent difference was found in the morphology of MI and MII triploids at the grow‐out stages. Discriminating formulae for triploid and diploid shrimp at grow‐out stage were developed and could be used to distinguish triploids from diploids based on morphological parameters. MI and MII triploids of shrimp have the potential to be used in aquaculture.     

The breeding potential and biochemical composition of triploid Manila clams, Tapes philippinarum Adams and Reeve

In 1989 and 1990. triploid Manila clam, Tapes philippinarum Adams and Reeve, seed were reared to 15-20 mm at the Fisheries Laboratory, Conwy, and planted out in the Menai Strait, North Wales. In each of the summers of 1992 and 1993, three of these batches, at 2, 3 or 4 years old, were returned to the laboratory to assess ploidy, size, spawning potential and biochemical composition. Percentage triploidy at this time was similar to that in the seed. After 6 and 8 weeks of warmwater conditioning. Only 45 out of l21 triploids (37%) were induced to spawn by thermal shock, with only one spawning as a male. By comparison, 75% of diploid clams spawned with a 1:1 ratio of males to females. Mean fecundity of triploids was significantly lower than that for diploids, at 0.497 compared with 1.54 million eggs per female. Compared with eggs from diploid females, eggs from triploids were larger and significantly fewer of them developed into D-larvae when fertilized by sperm from diploid males. Triploid clams were heavier and had a higher condition index and carbohydrate content than diploids of the same age, but lipid levels were similar. Potential advantages of producing and cultivating 100% triploid batches of Manila clam seed are discussed.     

Differences in metabolism of triploid and diploid Salmo trutta f. lacustris under acclimation conditions and after exposure to stress situations

Differences in metabolism (enzyme activities, metabolites) between diploid and triploid Salmo trutta f. lacustris were investigated under acclimation and stress conditions. Under acclimation conditions enzyme activities differed for 35% of the 27 investigated key regulatory enzymes and temperature optima for 23%. Muscle and liver metabolites related to energy metabolism and diagnostic indices of blood serum were similar, with exception of acetyl‐CoA being increased in triploids. Metabolic rate was lower and gill ventilation rate higher in triploids in comparison with diploids. During the tested stress situations (24 hr endurance swimming, 3 hr exposure to hypoxia in water with 32% oxygen saturation) muscle and liver glycogen decreased and serum and muscle lactate increased in both ploidy levels. Specifically, for triploids muscle adenylate energy charge and phosphocreatine levels decreased after endurance swimming and muscle and liver adenylate energy charge after exposure to hypoxia. Acetyl‐CoA increased in triploids during both stress situations. In summary, there existed differences in metabolism between the two ploidy levels and the energy metabolism of triploids was less balanced under stress.     

Studies on aneuploid pearl oyster (Pinctada martensii Dunker) produced by crossing triploid females and a diploid male following the inhibition of PB1

Viable aneuploid embryos of pearl oyster, Pinctada martensii Dunker, were produced by inhibiting the first polar body (PB1) with cytochalasin B treatment in eggs from triploids fertilized with haploid sperm. During the period of growth measurement, aneuploid showed the slowest growth compared with diploid and triploid groups. The body size and weight measurement data showed that there were no differences between aneuploids (as a group) and diploids in body size and weight (P>0.10), but that they were significantly different from triploids (P<0.01). There were no differences between aneuploids within diploid condition (2n±n) and diploids in SL (P>0.1), but significant differences in BW were found (P<0.05). Aneuploids within triploid condition (3n±n) were significantly smaller than triploids in BW (P<0.05), but not different from triploids in SL (P>0.05), and almost identical to diploids in both (P>0.1). These dada indicated that some aneuploids might be associated with growth retardation. Karyotype analysis revealed that there were metacentric, submetacentric or subtelocentric chromosomes losses or gains; aneuploid pearl oysters with the same chromosome numbers had different chromosome composition. Aneuploids are valuable research materials for genetic analysis.     

Survival of Diploid and Triploid Rhamdia quelen Juveniles Under Different Oxygen Concentrations

The objective of this study was to determinate the lethal concentration of dissolved oxygen (DO) over 96 hours of exposure (LC50–96h) for diploid and triploid jundia Rhamdia quelen juveniles. Diploid and triploid fish weighing approximately 4 g were subjected to DO concentrations varying between 0.4 and 1.3 mg O₂ L^?1; water temperature was maintained at 27?°C and pH at 6.3. The LC50–96h for diploids of Rhamdia quelen was 0.535 mg O₂ L^?1, while the value obtained for triploids was 6% greater. These results demonstrated that triploids of Rhamdia quelen juveniles have greater sensitivity to hypoxia compared to diploids.     

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

The purpose of this study was to investigate reproductive ability of backcross triploid koi (Cyprinus carpio L.) × goldfish (Carassius auratus L.) hybrids. These triploids have been obtained by crossing of F₁ hybrid females producing diploid eggs with males of parental species. Triploid hybrid females, when crossed with goldfish or koi males, produced mostly aneuploid fish with ploidy range from approximately 2.2n–3.2n with a mean value 2.5n; some fish in crosses of triploid females with koi males were tetraploid (4.0n). Since analysed fish had in their genomes one haploid set from parental males, the data indicate that triploid hybrid females mostly produced aneuploid eggs with ploidy range from approximately 1.2n–2.2n and a mean ploidy around 1.5n while some eggs were triploid. Triploid hybrid males were completely sterile and have not released any sperm after hormonal injection. Despite their low viability, some aneuploid fish obtained from triploid hybrid females were raised in indoor recirculating systems until the age of 2 years and their reproductive ability has been evaluated. One aneuploid female with ploidy 2.1n produced larvae with ploidy range from 2.9n to 3.4n with a mean ploidy of 3.1n when crossed with a koi male; about 60% of obtained larvae had ploidy from 3.0n to 3.2n. These data indicate that this female produced mostly eggs with unreduced ploidy level.     

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.