Muscle fibre growth dynamics in diploid and triploid rainbow trout

The effect of triploidy on muscle fibre growth was determined by comparing hyperplasia and hypertrophy of white muscle fibres in all-female, diploid and triploid rainbow trout Oncorhynchus mykiss (100–400 mm total length). Conventional morphometry and protein and DNA concentrations were used to assess muscle fibre hyperplasia and hypertrophy in white muscle samples derived from an anterio-dorsal location. Muscle fibre distributions were significantly different between triploids and diploids in trout <300 mm. The proportion of fibres <20 μm was higher in diploids than in triploids and the proportion of fibres in the 20–40 μm category was higher in triploids than in diploids. This indicates that the hyperplastic fibres of triploids are larger than those of diploids. Larger hyperplastic fibres in triploids are probably due to the combined effect of increased nuclear size in triploids and the relatively high nucleus: cell ratio observed in small muscle fibres. These larger fibres may be less favourable to cellular metabolic exchange because of their smaller surface area to volume ratios, and perhaps account for reduced viability and growth observed in triploids during early life stages. On the other hand, the lack of difference in the distribution of fibres <20 μm between diploids and triploids at larger body size ranges (301–400 mm) imply that triploid trout may have higher rates of new fibre recruitment and growth capacity at these sizes. There was no difference between diploid and triploid trout in the mean size of muscle fibres; however, the number of fibres per unit area was reduced by 10% in triploids. No differences were observed in protein or DNA concentrations in muscle tissues between the two genetic groups. Since triploid nuclei have 1·5 times more DNA than diploid nuclei, this deviation from the expected muscle DNA concentration (1·3–1·4 times more DNA in triploids when the 10% reduction in fibre density is considered) suggests that the number of nuclei per muscle fibre is reduced. In both diploids and triploids, mean fibre size increased with body length while fibre density decreased. Similarly, protein concentration in the muscle tissue increased and DNA concentration declined with increasing body length. Protein/DNA ratio was strongly and positively correlated with fibre size. These results demonstrate that changes in DNA and protein concentrations can be used to assess hyperplasia and hypertrophy in muscle tissues. However, the morphometric procedure provides better insight into muscle fibre growth as it enables the direct visualization and analysis of muscle fibre distribution patterns.     

Gonadal morphology of female diploid gynogenetic and triploid rainbow trout

Chromosome sets of fishes can be manipulated; this practice includes the production of triploid and gynogenetic salmonids. Such chromosomal modifications often result in abnormal ovarian development. In rainbow trout (RBT), triploid females have string-like gonads lacking significant developing oocytes and are suggested to be sterile due to the odd set of chromosomes disrupting oogenesis. Aberrant ovarian development is reported to occur in about 30% of gynogenetic females. It has been suggested that gynogenetic fish are more prone to expressing developmental abnormalities due to either increased homozygosity or to incomplete inactivation of the paternal chromatin. This investigation was done to compare the ovarian morphology of female triploid and induced gynogenetic diploid RBT. The objective was to determine whether the presence of supernumerary chromosomal fragments, potentially generated during the process of sperm genome inactivation, would result in abnormal gonadal development in gynogens comparable to that observed in triploid females. Gonadal morphology was observed and karyotypical analysis was completed on 21 gynogenetic fish. In 90% of the fish examined, the presence of chromosomal fragments was positively correlated with irregular ovarian development. The atypical gonadal morphology observed in the gynogens resembled triploid RBT ovarian morphology. The results of this investigation support the hypothesis that disruption of the normal diploid chromosomal complement alters germ cell development in gynogenetic female RBT due to the unbalanced nature of the genome. J. Exp. Zool. 286:505-512, 2000.     

Biochemical and morphological correlates of growth in diploid and triploid rainbow trout

Diploid and triploid rainbow trout contained the same amount of RNA per unit weight of white muscle tissue at a given growth rate. This finding, along with a previous finding that the protein content of triploid muscle tissues is not different from that of the diploids, indicates that triploid nuclei produce more RNA than their diploid counterparts. This increased production of RNA is probably due to the expression of the extra set of chromosomes in triploid nuclei. RNA-based growth correlates and liver somatic index ( I _H) were valid predictors of growth for both triploid and diploid fish. RNA concentration appeared to be better than RNA/DNA and RNA/protein in predicting growth.     

Comparison of reproductive development in triploid and diploid female rainbow trout Oncorhynchus mykiss

The diploid rainbow trout Oncorhynchus mykiss reached sexual maturity 3 years after hatching and its oogenesis underwent four stages, which were oogonia, primary oocyte, secondary oocyte and egg. Reproductive development and hormone changes of 4 to 35 month‐old female O. mykiss were investigated using histological and radioimmunoassay methods in order to provide a theoretical and practical basis for the use of triploid female O. mykiss. The oogonium of the triploid female could develop into the oocytes of the prophase with abortion occurring later; the oogonium was surrounded by stroma cells to form the oogonium cluster and the gonads showed a virilescent tendency when the oogonium clusters were gradually replaced by spermatogenic‐like cytocysts. After 13 months, amounts of gonadotropic hormone (GtH‐I, GtH‐II) and oestradiol (17β‐E₂) in triploid females were lower than in diploid fish at corresponding time periods, but the amounts of testosterone (T) increased consistently after 21 months and were more than in diploid fish in the corresponding time periods (P > 0·05). The infertility of triploid females resulted from meiosis failure, which caused developmental abortion of oocytes and oogonium formed cytocysts before the prophase oocytes. The cytocyst formation was due to the lack of the normal interaction of ovum and follicular cells, the development of follicular cells producing steroids were inhibited, the arylate path from T to 17β‐E₂ was interrupted, concentration of 17β‐E₂ decreased and concentration of T increased in the blood, the content of vitellogenin (Vg) decreased in the liver with a low 17β‐E₂ and high T caused to ovaries to show a tendency to be virilescent.     

Effects of progesterone and estradiol on the reproductive axis in immature diploid and triploid rainbow trout

In fish species, many studies demonstrated the crucial role of estradiol (E2) in the development of the reproductive axis, but progesterone (P) has been described mainly as a precursor steroid and no clear role by itself has been reported. Moreover, a cooperative effect of P (or another progestin) and E2 in fish has never been reported to our knowledge. In the present work, we investigated the effects of P, alone or in combination with E2, on the reproductive-axis of immature rainbow trout (Oncorhynchus mykiss). Liver vitellogenin and estradiol receptor (rtER) mRNA levels increased after E2 treatment, but were unchanged by P treatments as a reflection of peripheral action of steroids. In contrast, at the pituitary level, LH contents increased after E2 and/or P treatments. Focusing on the brain level, we confirmed a clear up regulation of rtER expression by E2 in sterile triploid females, and we also demonstrated a similar stimulating effect of P alone but no cooperative effect together with E2. In conclusion, our data demonstrate that in immature trout, prior to the beginning of the first reproductive cycle, unlike E2, P is able to stimulate the reproductive brain-pituitary axis without affecting vitellogenin synthesis in the liver.     

Production of androgenetic diploid rainbow trout

Haploid androgenesis was induced in rainbow trout (Salmo gairdneri) when eggs were irradiated with 60Co gamma radiation prior to fertilization. Diploidy was restored to the androgenetic haploid zygotes by suppression of first cleavage division using hydrostatic pressure. Peak survival in the androgenetic diploid lots (32.5-38.9 percent of control) occurred when a pressure shock of 9000 pounds per square inch lasting from one to three minutes was applied to the eggs 345 minutes post-fertilization. Chromosomal analysis confirmed diploidy in the androgenetic individuals and suggested that YY rainbow trout are viable to at least the "eyed stage" of development. Inheritance patterns at two loci confirmed all-paternal inheritance. The relatively high yields of completely homozygous androgenetic rainbow trout and the potential for the use of androgenesis in the production of inbred lines and in genetic studies indicate that androgenesis may become a valuable tool in fish research and breeding.     

The critical swimming velocity of diploid and triploid brook trout

No significant difference was found in the critical swimming velocity of diploid and triploid brook trout Salvelinus fontinalis , suggesting that the triploid state is not associated with a reduced aerobic capacity.     

Sexual maturation in triploid rainbow trout, Salmo gairdneri Richardson

This paper compares some morphological and endocrinological characteristics of diploid and triploid rainbow trout.
Significant differences were found between diploid and triploid females in GSI, condition factor, gut weight, liver weight and percentage dress-out, and between diploid and triploid males in GSI, condition factor and gut weight.
Diploid females had large, well-developed ovaries containing yolk-filled secondary oocytes whereas the triploids had only string-like ovaries containing nests of oogonia. No primary oocytes were present.
All the diploid males produced copious quantities of milt but it was possible to express a thin, watery milt containing motile spermatozoa from only two of the 12 triploid males. Testes weights in triploids were similar to those of diploids but, while the diploid testes were packed with spermatozoa, those of the triploids consisted mainly of spermatocytes and spermatids with few spermatozoa present. Measurements of the heads of spermatozoa revealed that those from triploids were larger and had a wider size range than those from diploids.
Levels of testosterone and 11-ketotestosterone in triploid and diploid males were not significantly different. However, levels of testosterone and 17β-oestradiol in diploid females were considerably higher than those of triploid females.     

The feeding response of diploid and triploid Atlantic salmon and brook trout

Pair and group experiments were conducted to determine whether differences exist in feeding success between juvenile diploid and triploid salmonids in a competitive situation. In the pair experiments, 22 pairs (one diploid and one triploid) of size-matched Quebec-strain brook trout Salvelinus fontinalis (7·2–46·3 g) were fed an unlimited number of pellets three times a day for 5 days. Dominance was assigned to the fish which ate the most pellets within each pair. In the group experiments, groups of three diploid and three triploid size-matched fish were fed a restricted ration three times a day for 5 days. Hierarchical rank within the group was assigned based on the number of pellets consumed by each fish. The group experiment was repeated 10 times with Atlantic salmon Salmo salar (5·1–62·7 g), Quebec-strain brook trout (11·8-110·8 g), and large UNB-strain brook trout (18·2–33·0 g), and 12 times with smaller UNB-strain brook trout (0·6–2·0 g). A statistically significant difference in rank between ploidies was found only for the smaller UNB-strain brook trout in the group experiments, with diploids dominant over triploids. This suggests that there may be a difference in competitive feeding success between diploid and triploid brook trout early in development, but that this difference diminishes as the fish grow.     

Gonadal sex reversal in triploid rainbow trout (Oncorhynchus mykiss).

Sex determination in salmonids is primarily governed by sex chromosomes; however, phenotypic expression and successful development of the gonads may be influenced by additional factors. Exposure to exogenous steroids during the critical period of gonadal differentiation will reverse the expected phenotypic sex of both female and male trout. Triploidy, a viable condition in rainbow trout (RBT), alters the degree of gonadal development in a gender-specific manner. Males produce testes with similar morphology and function as diploid fish, but females produce underdeveloped ovaries devoid of growing oocytes. One possible explanation for this observed gender difference is that the timing of meiotic initiation may influence ovarian/testicular development in triploid RBT. To determine whether the early entrance of germ cells into meiosis results in the lack of ovarian development in triploid females, the objective of this study was to sex-reverse genotypic triploid female RBT (XXX) into phenotypic males and genotypic triploid male RBT (XXY) into phenotypic females. Male fish were exposed to estradiol-17beta (E(2)) and females were exposed to the non-aromatizable androgen 17alpha-methyldihydrotestosterone (MDHT). Over 90% of the male fish treated with exogenous E(2) developed gonadal structures indistinguishable from the gonads of triploid females. Triploid female RBT treated with MDHT developed testes; however, not all fish treated with this androgen were completely sex reversed. The results of this investigation are consistent with the hypothesis that the failure of ovarian development in triploid RBT is due to the early onset of meiosis and does not appear to be due to genotypic sex. J. Exp. Zool. 284:466-472, 1999.     

The physiological response of diploid and triploid brook trout to exhaustive exercise

Using triploidy as an experimental model, we examined whether cell size limits the post-exercise recovery process in fish. Because triploids generally possess larger cells, which could affect many physiological and biochemical processes, we hypothesized that triploids would take longer to recover from exhaustive exercise compared to diploids. To test this, we measured plasma lactate, glucose and osmolality, and white muscle energy stores (glycogen, phosphocreatine and ATP) and lactate before and immediately following exhaustive exercise and during recovery at 2 and 4 h post-exercise. In addition, oxygen consumption and ammonia excretion rates were determined before and after exhaustive exercise. Overall, diploid and triploid brook trout showed similar metabolic responses exercise, but plasma osmolality, white muscle lactate, white muscle ATP and post-exercise oxygen consumption rates recovered earlier in triploids compared to diploids. The results of this study suggest that the characteristic larger cell size of triploidy does not limit the physiological response to, or recovery from, exhaustive exercise.     

Use of diploid and triploid trout erythrocytes as internal standards in flow cytometry.

DNA content determination requires the use of standards. Vindelov has shown the need to use two standards. Chicken and trout erythrocytes are commonly used, but they are not ideal standards. On the one hand, their DNA contents rarely frame the studied sample DNA content, and, on the other hand, as their base compositions are different in terms of A + T/G + C, their relative indices change according to the stains used. Use of triploid trout erythrocytes instead of chicken erythrocytes allows elimination of these two drawbacks; however, diploid trout must be differentiated from triploid trout. The present paper shows that an anatomic malformation is found with the triploid trout and so justifies the use of paired diploid and triploid trout as standards to measure nuclear DNA content.     

Amitosis‐like nuclear division in erythrocytes of triploid rainbow trout Oncorhynchus mykiss

This work shows that the atypical erythrocytes in triploid rainbow trout Oncorhynchus mykiss were morphologically similar to those of toads. The nuclei of the cells can be bell‐shaped, constricted or irregular. It is presumed that such nuclear division is probably amitosis.     

Influence of growth rate on white muscle dynamics in rainbow trout and brook trout

In trout, fast growth stimulated white muscle fibre hypertrophy ( P 0·001) and hyperplasia ( P <0·01) in outer fibres but not in deep fibres. Glycogen was most prevalent in outer fibres ( P <0·01) and in brook trout ( P <0·01) that on average had three to four times larger fibres than rainbow trout.     

Effects of exogenous estradiol-17β on early growth and gonadal development of diploid and triploid female rainbow trout (Oncorhyncus mykiss)

Triploidy is a viable condition in teleosts. However, in many salmonids, the triploid condition in the female results in sterility as gametogenesis appears to be disrupted. Although the underlying mechanisms regulating the gonadal development of teleosts have not been clearly elucidated, the reversal of phenotypic sex by the administration of the appropriate exogenous steroid during early development supports the argument that gonadal steroids play a pivotal role in sexual differentiation and subsequent gonad development in these fish. To determine whether the failure of normal ovarian development in triploid female rainbow trout (Oncorhynchus mykiss) is due to an absence or reduction of endogenous sex steroids, ovarian morphology was compared between diploid and triploid juvenile rainbow trout treated with exogenous estradiol-17β (E₂). The ovaries of both untreated and E₂ treated diploid fish, at 145 days post-fertilization, contained synchronously developing oocytes in the perinucleolar stage, whereas ovaries from untreated and estradiol-treated triploid fish of the same age were considerably smaller and devoid of developing oocytes. No differences in the ovaries of triploid untreated fish and triploid fish treated with E₂ were observed. It is reported that exposure to exogenous E₂ during the period of gonadal differentiation is not sufficient to induce oocyte development in triploid rainbow trout. © 1996 Wiley-Liss, Inc.     

Comparison of growth performance,biochemical and fatty acid compositions between all‐female diploid and triploid rainbow trout Oncorhynchus mykiss (Walbaum, 1792)

This study compares the performance (in terms of survival and growth), biochemical and fatty acid compositions among all female diploid (AFD) and triploid (AFT) rainbow trout (Oncorhynchus mykiss). AFD and AFT fish with mean initial weights of 1,040.1 ± 1.3 and 1,039.7 ± 1.6 g, respectively, were reared and fed from March to August with a commercially extruded trout feed in a commercial freshwater fishfarm located in the Black Sea region (Samsun, Turkey). Survival was reduced throughout the experimental period in the AFT group with increasing water temperatures. At the end of the experiment, survival rates were 98.57 ± 1.43% and 82.38 ± 7.39% for the AFD and AFT groups, respectively. The AFD group showed significantly better growth performances in terms of weight gain, feed conversion rate (FCR), relative growth rate (RGR) and specific growth rate (SGR) than the AFT group (p < .05). Significantly less protein and greater fat content were also observed in the AFT group (p < .05). There was no significant difference between groups for fatty acid composition in meat, except for stearic acid (SA, C18:0) and docosahexaenoic acid (DHA, 22:6 n‐3). While the greatest SA concentration was in the AFT group, the greatest DHA concentration was observed in the AFD group (p < .05). The results indicate that female triploid rainbow trout are more susceptible to suboptimal environmental conditions (especially to higher water temperatures) than female diploids. Although poor triploid performance was observed in this study, relative productivity might be enhanced by rearing triploids in optimal environmental conditions.     

Genome‐wide methylation study of diploid and triploid brown trout (Salmo trutta L.)

The induction of triploidization in fish is a very common practice in aquaculture. Although triploidization has been applied successfully in many salmonid species, little is known about the epigenetic mechanisms implicated in the maintenance of the normal functions of the new polyploid genome. By means of methylation‐sensitive amplified polymorphism (MSAP) techniques, genome‐wide methylation changes associated with triploidization were assessed in DNA samples obtained from diploid and triploid siblings of brown trout (Salmo trutta). Simple comparative body measurements showed that the triploid trout used in the study were statistically bigger, however, not heavier than their diploid counterparts. The statistical analysis of the MSAP data showed no significant differences between diploid and triploid brown trout in respect to brain, gill, heart, liver, kidney or muscle samples. Nonetheless, local analysis pointed to the possibility of differences in connection with concrete loci. This is the first study that has investigated DNA methylation alterations associated with triploidization in brown trout. Our results set the basis for new studies to be undertaken and provide a new approach concerning triploidization effects of the salmonid genome while also contributing to the better understanding of the genome‐wide methylation processes.