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of white muscle fibres in normal-sex-ratio fish increased ... development, ploidy, sex-reversed fish. ..... of possible diameters, fixing the maximal diameter was.
1991

The Journal of Experimental Biology 202, 1991–2016 (1999) Printed in Great Britain © The Company of Biologists Limited 1999 JEB1947

MUSCLE GROWTH AND DEVELOPMENT IN NORMAL-SEX-RATIO AND ALLFEMALE DIPLOID AND TRIPLOID ATLANTIC SALMON IAN A. JOHNSTON1,*, GILLIAN STRUGNELL1, MARTI L. MCCRACKEN2 AND RAY JOHNSTONE3 Marine Laboratory, School of Environmental and Evolutionary Biology, 2School of Mathematics and Computational Sciences, University of St Andrews, St Andrews, Fife KY16 8LB, Scotland and 3Fisheries Research Service, Marine Laboratory, SOAEFD, Aberdeen, Scotland 1Gatty

*e-mail: [email protected]; http://www.st-and.ac.uk/~fmrg/

Accepted 24 April; published on WWW 7 July 1999 Summary respectively, for all-female fish. The probability density Muscle development and growth were investigated in function of muscle fibre diameters in each fish was diploid populations of normal-sex-ratio and all-female estimated using non-parametric smoothing techniques, and Atlantic salmon (Salmo salar L.) and their triploid the mean densities for diploids (fD) and triploids (fT) were counterparts produced by high-pressure treatment. −1 calculated. The peak fibre diameter was approximately Somites were formed at the rate of 6 h in both diploids and triploids at 6 °C. The rostral-to-caudal development of 20 µm in all age classes, irrespective of ploidy. Distinct myotubes, myofibrils and acetylcholinesterase staining at bimodal distributions of muscle fibre diameter were the myosepta was slightly more advanced in triploid than evident in all groups 775 days and 839 days post-hatching, in diploid fish, although the differences were smaller than reflecting seasonal cycles of fibre recruitment. fD and fT among individual families. The c-met receptor tyrosine were compared using a non-parametric bootstrap kinase was used as a molecular marker for the satellite cells technique and the reference band representing the nullinvolved in postembryonic muscle growth. Satellite cell hypothesis indicated that there was no difference with nuclei comprised 17.5 % of total myonuclei in smolts and ploidy. Reference bands for normal-sex-ratio fish at 315 they were 24 % more abundant in diploid than in triploid days and 470 days indicated that diploids had a higher fish. Cells expressing the myogenic regulatory factor myfpercentage of smaller-diameter fibres and that triploid 6, a marker of satellite cells committed to differentiation, distributions had a thicker right-hand tail. Similar represented 14.8 % of total myonuclei in diploids and differences in fD and fT of muscle fibre diameters were 12.5 % in triploids. At ambient temperatures, the number found for all-female fish, although the statistical evidence of white muscle fibres in normal-sex-ratio fish increased was less strong. Reference bands indicated differences in more than 30-fold between the alevin and smolt stages, and the middle range of the distributions of muscle fibre approximately 3.5-fold further during the first year of diameter in fish 620–775 days post-hatch, with triploids seawater growth. The rate of muscle fibre recruitment in having a thicker right-hand tail. Thus, a lower density of seawater stages was significantly greater in diploid satellite cells was associated with reduced rates of fibre than in triploid fish, reaching 1162 fibres day−1 and recruitment but a compensatory increase in muscle fibre 608 fibres day−1, respectively, in all-female groups 800 days hypertrophy in triploid compared with diploid fish. post-hatching. For 42 cm fork-length fish, there were approximately one-third more muscle fibres per myotome Key words: Atlantic salmon, Salmo salar, muscle, growth, development, ploidy, sex-reversed fish. in diploid than in triploid groups, 649 878 and 413 619,

Introduction Polyploidy has been a significant factor in the evolution of salmonids (Allendorf and Thorgaard, 1984; Johnson et al., 1987). Low frequencies of triploid salmonids occur in natural populations (Thorgaard and Gall, 1979), and triploidy can be readily induced experimentally by either heat or pressure treatment (Benfey, 1991). There are fewer, but larger, cells in most organs and tissues of triploid fish, and it has long been suspected that this may have far-reaching consequences for their development and biology (Small and Benfey, 1987;

Benfey, 1991). For example, the lower numbers of brain and sensory cells in triploids is correlated with marked changes in behaviour, including a decreased sensitivity to light and sound (Aliah et al., 1990) and reduced aggressiveness relative to diploids (Benfey, 1991). Although mitotic cell divisions proceed normally in triploids, the presence of the third chromosome means that balanced chromosome constitutions cannot be established at first meiosis. Triploids are therefore functionally sterile. In fish farming, maturation essentially

1992 I. A. JOHNSTON AND OTHERS marks the end of the useful period of rearing and, if economic yields are to be maximised, fish must be sold before the deteriorative changes associated with maturation lower their market value. Salmon farmers became interested in the use of triploid fish for sterility reasons (for a review, see Ihssen et al., 1990). Because of the potential genetic introgression threat posed by the escape of diploids from fish farms, salmonid stock managers are also interested in the sterility of triploids (for a recent review, see Youngson et al., 1997). If triploid salmon could be reared as economically as diploids, then the introgression threat to wild populations would be minimised at little cost to farmers. Although male triploids are functionally sterile, they produce near-normal levels of circulating androgens at spawning time and therefore suffer the normal deteriorative changes associated with maturation (Lincoln and Scott, 1984). Female triploids, because of differences in the architecture of the germinal and endocrine tissues, are both functionally and hormonally sterile (Sumpter et al., 1984). Only triploid females would therefore be of use in aquaculture. Sex in fish, although determined by the presence of a Y male-inducing chromosome, is extremely susceptible to manipulation. The induction of functional testes in genetic females (XX) is therefore straightforward, involving the addition of small amounts of hormones to the feed or rearing water (Johnstone and Youngson, 1984). These ‘inverted females’ (sex-reversed males) can be used to fertilise normal eggs (XX) to produce all-female offspring from which the Y chromosome has been eliminated. The effects of triploidy on growth are variable and probably depend on the induction method, husbandry practices and the stage of the life cycle being compared (Thorgaard, 1986). Several studies have reported that triploid fish grow more slowly than diploids when reared under communal conditions, which may be related to decreased aggressiveness and/or an increased susceptibility to stress. However, triploid Atlantic salmon often show growth performance as good as or better than that of diploids when reared separately (Carter et al., 1994; Johnstone et al., 1991). Somatic growth is closely related to that of the muscle tissue, which comprises approximately 65 % of the body mass (Weatherley et al., 1979). In Atlantic salmon, distinct germinal zones of myoblasts are present at the dorsal and ventral apices of the myotome during the yolk-sac stages (Higgins and Thorpe, 1990; Johnston and McLay, 1997). By first feeding, there are approximately 10 000 white muscle fibres per myotome, and at this stage small-diameter muscle fibres begin forming throughout the myotome on the surface of existing fibres (Higgins and Thorpe, 1990; Johnston and McLay, 1997). Surprisingly, given the importance of Salmo salar as a food fish, there have been no systematic studies of muscle recruitment during the seawater stages of the life cycle, although it is known that there are in excess of 1 million muscle fibres per myotome after two sea-winters of growth (Johnston, 1999). Muscle is a post-mitotic tissue, and post-embryonic growth

involves the satellite cell population, which proliferates to provide a source of nuclei for fibre recruitment and hypertrophy (Veggetti et al., 1990; Koumans et al., 1991; Johnston et al., 1995, 1998). It has been reported that the yield of mononuclear cells per gram of muscle tissue is significantly higher in primary cell cultures from diploid than it is from triploid rainbow trout (Oncorhynchus mykiss) (Greenlee et al., 1995), suggesting that there may be more satellite cells in diploids which, in turn, would be predicted to affect muscle growth characteristics. However, in rainbow trout greater than 3 cm in fork length, Suresh and Sheehan (1998) found that the frequency histograms of muscle fibre diameter were similar in diploid and triploid fish. These authors indirectly estimated that there were 10 % fewer muscle fibres per unit cross-sectional area in triploid than in diploid individuals. The objective of the present study was to provide a comprehensive description of muscle growth in diploid and triploid Atlantic salmon (Salmo salar L.) using both normalsex-ratio and all-female populations. Specifically, we wished to test the hypotheses that ploidy influences the relative timing of embryonic myogenesis, the number of satellite cells per myotome and the rate of muscle fibre recruitment. Nonparametric smoothing techniques were applied in a novel approach to investigating the density distribution of muscle fibre diameter, and hence hypertrophic growth, at different stages of the life cycle. Recently, the c-met tyrosine kinase receptor has been identified as a molecular marker of satellite cells in the mouse (Cornelison and Wold, 1997). The MyoD family of myogenic regulatory factors (MyoD, myogenin, myf-5 and myf-6) plays an important role in muscle differentiation and the factors are expressed in proliferating satellite cells (Grounds et al., 1992; Yablonka-Reuveni and Rivera, 1994; Cornelison and Wold, 1997). In the present study, the use of antibodies to c-met and myf-6 proteins to estimate the number of satellite cells in salmon smolts is also reported. Materials and methods Experimental stocks Three experimental series were conducted. In the first series, beginning in 1994, the eggs from two females taken from a fish farm were pooled and fertilised with either the milt from a normal farmed male, thus producing a population with a normal sex ratio (NSR), or with the milt from a sex-inverted female, thus producing an all-female population (AF; for experimental details, see Johnstone and MacLachlan, 1994). Shortly after fertilization, a proportion of both egg batches was made triploid by high-pressure treatment (5 min at 6.9×104 kPa) (Johnstone and Stet, 1995), thereby generating four comparative groups: diploid NSR, diploid AF, triploid NSR and triploid AF. This series of fish was reared for a prolonged period at the Fisheries Research Service salmonrearing facility at Aultbea, Wester Ross, Scotland, at ambient, i.e. fluctuating, temperature. Fish were weaned onto dry proprietary feed when the yolk was approximately 80 %

Salmon muscle growth 1993 25

series as monitored by light microscopic analysis of muscle nucleoli (diploids had one or two and triploids one, two or three per nucleus) or, in larger fish, by flow-cytometric analysis of erythrocytes.

S1

Temperature (°C)

20 15

SWT

10 5 FF 0 0

100 200 300 400 500 600 700 800 Age post-hatch (days)

Fig. 1. Water temperatures recorded during 1994–1997 at the Altbea fish cultivation unit, West Rosshire, Scotland. The filled symbols represent the temperatures for the freshwater stages (triangles, embryonic; circles, post-hatch) and the open symbols for the saltwater stages of the Atlantic salmon (Salmo salar L.) families studied. Although the temperature was recorded daily for clarity, only the data for approximately every tenth day are shown. FF corresponds to first feeding, S1 to the selection of fish in the upper growth mode during summer 1995 and SWT to the transfer of smolts to saltwater tanks.

exhausted (first feeding). S1 parr destined to become smolts after 1 year in fresh water were selected in September 1995. Fish were transferred to tanks at a uniform stocking density and fed with automatic feeders. All fish feeds were supplied by Trouw Aquaculture Ltd. Fry were initially fed unpigmented dry pellets 0.3–0.8 mm diameter at the rate of 3 % kg−1 biomass day−1. Pellet size was increased with growth, and feeding rates varied from 2 to 2.55 % kg−1 biomass day−1 during the summer months to 1 % kg−1 biomass day−1 in the winter. Fish were transferred to sea water in April 1996 and reared in replicated tanks at comparable stocking densities until May 1997. The temperature was recorded daily throughout the experiment. The minimum and maximum temperatures experienced by freshwater stages were 1.7 °C and 21 °C, respectively, compared with 8 °C and 14 °C for seawater fish (Fig. 1). In the second series of experiments, started in 1995, eggs from two wild salmon were fertilized with the milt from either an NSR farmed male or a sex-inverted female to produce two families of NSR and AF offspring. Approximately half the eggs were made triploid as before, and both groups were incubated at a constant temperature of 6±0.2 °C until they hatched. In the third experimental series (in 1996), the eggs from six female wild salmon were fertilized with the milt from one of two farmed males to produce six unique families of NSR fish. Diploid and triploid (prepared by pressure treatment as described above) groups were reared until first feeding at a constant temperature of 6±0.2 °C at the SOAEFD hatchery facility at Almondbank, Perthshire, Scotland. Using the protocol of Johnstone and Stet (1995), triploidisation success was 100 % in all three experimental

Embryonic development Embryos from each family were sampled daily until the end of somitogenesis and every 2 or 3 days thereafter. Eggs were dechorionated using fine forceps, and the embryos were removed without damage. From each sample, at least six live triploid and six live diploid embryos were examined from each family using a stereo microscope under both bright- and darkfield illumination. Once movements had started, the embryos were anaesthetized in a 1:5000 (m/v) solution of bicarbonatebuffered MS222 (ethyl m-aminobenzoate). The somite stage and the appearance of particular organs and tissue types were noted. Approximately 10 embryos from each group were fixed in each of the following fixatives (A) Bouin’s fluid and (B) 4 % (m/v) paraformaldehyde in 0.12 mmol l−1 phosphate-buffered saline (PBS) for subsequent histological and histochemical analysis, respectively. Embryos fixed in Bouin’s fluid were embedded in wax, and 7 µm serial sagittal sections were cut and stained with haematoxylin–eosin. The most posterior somite containing myotubes and myofibrils was scored for each embryo along with the somite stage. The appearance of functional endplates at the myosepta was investigated by staining embryos for acetylcholinesterase activity. Embryos were incubated in the dark for 3–5 h at 4 °C in a solution containing (in mmol l−1): copper sulphate, 3; potassium ferricyanide, 0.5; maleate buffer, 100; acetylthiocholine, 1.7. The staining reaction was stopped by rinsing several times in PBS, and embryos were mounted in glycerol under glass coverslips supported by silicone grease at each corner and examined using Nomarski differential interference (DIC) optics with a Leitz DRM Systems microscope. Studies of muscle growth Fish were sampled 46 and 57 days after fertilization (embryo stages), at hatching and at first feeding, and fixed in Bouin’s fluid prior to processing for wax histology. Following first feeding, muscle growth was assessed using frozen sections to avoid problems associated with shrinkage. A shrinkage correction factor was applied to fibre size measurements of the earlier stages on samples fixed in Bouin’s fluid. To quantify muscle cellularity, a cross section 3–5 mm thick was cut at the level of the pelvic fin insertions and photographed against graph paper. The cross section from one half of the body was divided into 3–10 labelled blocks depending on the size of the fish. Blocks were mounted on cork strips and frozen in 2methyl butane cooled to near its freezing point (−159 °C) in liquid nitrogen. Samples were wrapped in tin-foil to avoid desiccation and stored in a liquid nitrogen refrigerator until they could be processed. Frozen sections, 10 µm thick, were cut, air-dried and stained with the nuclear stain Scarba Red. The outlines of muscle fibres and the total cross-sectional area of muscle tissue were digitized using an image-analysis

1994 I. A. JOHNSTON AND OTHERS system, and the equivalent muscle fibre diameters were calculated (Kontron Electronics, Basel, and ScanBeam, Denmark). The estimated value of the total number of muscle fibres per myotome was plotted against the cumulative number of fibres sampled until a stable estimate was obtained. Between 800 and 1200 muscle fibres were measured per fish, representing fields from all areas of the myotomal cross section. Immunohistochemistry Transversely cut 7 µm thick frozen sections of muscle were mounted on poly-L-lysine-coated glass slides and air-dried for approximately 1 h. Sections were fixed for 10 min in 4 % (m/v) paraformaldehyde in PBS, washed three times for 3 min in PBS, blotted and placed in acetone for 10 min and finally airdried for 10 min. Prior to immunohistochemistry, sections were rehydrated in 1 % (v/v) Triton X-100 and 1 % (m/v) bovine serum albumin (BSA) (Sigma Chemicals, Poole, UK) in PBS. Background peroxidase activity was reduced by incubating the sections in 0.5 % (v/v) hydrogen peroxide for 10 min, and nonspecific binding sites were blocked for 15 min with a solution containing 4 % (m/v) normal goat’s serum, 1 % (v/v) Triton X100 and 1 % (m/v) BSA in PBS. Sections were incubated overnight in the primary antibody at 4 °C. Primary antibodies and dilutions used were as follows: rabbit anti-m-met (Santa Cruz) at 1:100, which stains the c-met tyrosine kinase receptor (Cornelison and Wold, 1997); and rabbit anti-myf 6 at 1:100 (Santa Cruz). After three washes in PBS, sections were incubated for 1 h in the secondary antibody biotinylated goat anti-rabbit IgG (Sigma Chemicals, Poole, UK) at a dilution of 1:20 in 1 % (v/v) Triton X-100, 1 % (m/v) BSA in PBS. Two control incubations were carried out omitting either the primary or the secondary antibody. Sections were washed three times in PBS and incubated for 1 h in a 1:20 dilution of extraAvidin peroxidase (Sigma Chemicals, Poole, UK) in 1 % (v/v) Triton X-100, 1 % (m/v) BSA in PBS. Peroxidase activity was developed using 3-amino-9-ethylcarbazole, which gives a red insoluble end-product. Duplicate sections were counterstained in Mayer’s haematoxylin to visualise total myonuclei. Slides were mounted under coverslips using gelatine and stored in the dark. Counts of the number of c-met/myf-6-positive cells per nucleus were made from at least six fields of approximately 50 muscle fibres per field in each fish using an image-analysis system (Scanbeam Ltd, Denmark) and related to the total cross-sectional area of muscle. Electron microscopy Small bundles of white muscle fibres were isolated from the dorsal epaxial myotomes of NSR salmon at first feeding and fixed overnight in 2.5 % (v/v) glutaraldehyde, 2.5 % (m/v) paraformaldehyde in 100 mmol l−1 sodium cacodylate buffer, pH 7.4 at 4 °C. Samples were processed for electron microscopy as described previously (Johnston et al., 1995). Sagittal ultrathin sections of muscle fibres were cut, stained with lead citrate and uranyl acetate, and viewed with a Philips 301 transmission electron microscope. The dimensions of

satellite cells were measured from photomicrographs at a magnification of 5000 times. Statistics The relationship between developmental characters and age or somite interval was fitted by linear least-squares regression. A two-way general linear model (GLM) analysis of covariance (ANCOVA) was used to test for differences in development, with ploidy and family as fixed effects and age or somite interval as a covariate (SPSS Statistical Software, SPSS Inc., USA). The somite interval (the mean time to make a somite) was calculated by least-squares regression and is equivalent to the somite stage of the embryo up until the end of somite formation. Growth variables such as body mass, the total crosssectional area of muscle and the number of fibres per myotome were plotted against either age post-hatch or fork length. Data were fitted by least-squares regression using a second-order polynomial and, in cases where a rate variable was required, the resulting curve was differentiated (Mathematica software, Wolfram Research Inc., USA). The hypothesis that there was a single population underlying the fitted curves was tested using multiple regression analyses (Zar, 1984). The effects of ploidy on muscle cellularity variables was tested using ANCOVA with fork length as a covariate (SPSS statistical software, SPSS Inc., USA). Satellite cell densities were compared using a one-way analysis of variance (ANOVA). The data for NSR and AF fish were analysed in separate ANCOVAs since these groups were half-siblings and therefore differed in genetic background. To evaluate and compare distributions of muscle fibre diameter, smooth non-parametric estimates of the probability density functions (PDFs) were constructed using the kernel approach (Silverman, 1986) within the S-Plus computing environment utilising the sm library (Bowman and Azzalini, 1997). The kernel method uses a smooth kernel function that is itself a PDF, such as the normal curve, as the basic building block. These smooth functions are centred directly over each observation, resulting in a smooth estimate of density while preserving the value of the realisation. The variance of the kernel function is controlled by the smoothing parameter h. The kernel estimator used was of the form: n

fˆ(y) =

冱 w(y − y ;h) , i

(1)

i=1

where fˆ is the estimated probability density function, yi is the ith observation from the list of n and w is the kernel function. For simplicity when comparing groups, we sampled an equal number n of fish in each group, and we sampled an equal number m of fibres within each fish. We then estimated the PDF for each fish using the normal optimal smoothing parameter (Bowman and Azzalini, 1997). Sample sizes were 800–1000 per fish, and smoothing parameters were within the range 0.13–0.19. The mean PDFs for diploid (fD) and triploid (fT) groups were then estimated using the diameters pooled

Probability density function

Salmon muscle growth 1995 0.01

A

0 0

0.025

50

100

150

200

50 100 150 Muscle fibre diameter (µm)

200

B

0 0

Fig. 2. A comparison of the histogram (A) and mean probability density function (B) for muscle fibre diameters from a triploid normal-sex-ratio Atlantic salmon 775 days post-hatch.

over group. Since it is advantageous to use a common smoothing parameter when comparing densities (Bowman and Azzalina, 1997), fD and fT were estimated with h equal to the mean of the normal optimal smoothing parameters over fish within an age class. To restrict diameters to positive values, we estimated density functions of the natural logarithm of diameter and then transformed back to the original scale. Furthermore, we fixed the maximal diameter within an age class at 110 % of the maximum diameter in the age class. Since the fit of the right-hand tail is dependent on the right endpoint of possible diameters, fixing the maximal diameter was required for consistency when comparing tail percentages. Fig. 2 shows a histogram and mean density estimate of fibre diameters for an NSR fish at 775 days post-hatch as an example. Approximating confidence bands for fD and fT would be the next logical step; however, the kernel estimator is biased, and the form of this bias as well as the form of the variance of fˆ makes approximating confidence bands computationally very complicated (Bowman and Azzalina, 1997). An alternative to a confidence band is a variability band. A variability band represents only the variability of an estimate, but it is still very helpful in strengthening the evidence of a structure characteristic, such as a right-hand tail or a bimodal distribution. The variability band of fˆ was approximated using bootstrap techniques. The hierarchical structure of the data (the observational unit was fish, with fibres being subunits) results in two sources of variation: variation among fish and variation within fish. It is important to take into account these two sources of variation when drawing bootstrap samples. One strategy is to sample randomly n fish with replacement and then to sample randomly, with or without replacement, m fibres within a fish. For a moderate sample size of, for example, 10 fish, sampling fibres without replacement is preferred (Davison and Hinkley, 1997). Since we had a small sample size of fish, we selected fibres within a fish using a smooth bootstrap sample as described by Silverman (1986) and Davison and Hinkley (1997). For each bootstrap sample, the mean density was estimated

and the area between this estimate and fˆD or fˆT was shaded. The final shaded area represents the maximal area created by the 100 bootstrap estimates of density and is referred to as the variability band. If the structural characteristic of interest is also common in the area, there is evidence that the characteristic is genuine. For example, if after 100 bootstrap replications the area did not have the characteristics of a bimodal distribution apparent in the estimate of mean density, we concluded that there was insufficient evidence to support a bimodal distribution. The next step of the analysis was to test the null hypothesis that fD=fT over all diameters. To test this hypothesis, nonparametric bootstrap test procedures were used. First, the Kolmogorov–Smirnov two-sample test statistic Dmn was calculated: Dmn =

max |

| | Smn(y) − Tmn(y) | , y| |

(2)

where Smn(y) and Tmn(y) are the empirical distribution functions of the m↔n fibre diameters y for each group (Gibbons, 1971). To approximate the P-value of this statistic, the bootstrap samples must be drawn from the distribution that satisfies the relevant null hypothesis. This was accomplished by resampling in three stages: (1) within each group, n fish were sampled with replacement (2) ignoring original groupings, group labels were randomly assigned such that there were n fish in each group and (3) a smooth bootstrap sample of m fibres for each fish was generated. After 100 bootstrap replications, the P-value was approximated by: P=

1 + #[D*艌D] , R+1

(3)

where #[D*⭓D] is the number of D*⭓D, and using the results D1*, ....., DR* from the R bootstrap samples (Davison and Hinkley, 1997). Since the sample size of fish within a group is small, 4–5 fish, the null hypothesis will probably not be rejected if a significance level of 0.05 is required unless a large real difference exists. Since significance level and power are related, given a fixed sample size (Zar, 1996), the critical significance level was increased to 0.1 as suggested by Steel and Torrie (1980) to increase the power of the test. This Kolmogorov–Smirnov-based bootstrap test is a global test, and a significant result strengthens the evidence that the densities are different, but the test statistic alone provides few clues to where the differences might occur. To supplement this test, the two density curves were compared graphically. First fˆD and fˆT were plotted. If the null hypothesis was true, it would be natural to ignore group labels and pool fibre diameters over all fish sampled in an age class when estimating the average density (fˆD+T) for an age class. To assess where the differences between fˆD and fˆT can be attributed to a genuine difference in structure and where they can be attributed to random variation, a variability band for fˆD+T was constructed. The region where fˆD and fˆT lie outside this band suggests a major difference

1996 I. A. JOHNSTON AND OTHERS between the densities. Since this band provides a means of evaluating the differences between fˆD and fˆT, it is referred to as a reference band. The final stage of evaluating differences between groups was to compare the values of specific percentiles of the estimated fibre densities for each fish. The Wilcoxon two-sample nonparametric test was used to test whether the median value of the specified percentile was equivalent between groups. Specified percentiles were the fifth, tenth, fiftieth, ninety-fifth and ninety-ninth percentiles. Since we were not taking into account the variance of the percentile estimate for each fish, and we were making multiple comparisons, the reported Pvalues should be interpreted with caution. Results Embryonic development The unsegmented paraxial mesoderm started to segment approximately 17 days post-fertilization at 6 °C, producing

Fig. 3. (A) A sagittal section of a normal-sexratio diploid salmon embryo with epithelial somites (es) sampled 22 days post-fertilization at the 28-somite stage and stained with haematoxoylin–eosin. Somites 18–23, counting from the head, are illustrated. Arrows indicate cells actively involved in mitosis. Scale bar, 25 µm. (B) Sagittal section of a normal-sex-ratio triploid salmon embryo at the 46-somite stage stained with haematoxoylin–eosin. Long arrows indicate mononuclear myotubes at the position of the horizontal septum. The short arrow indicates a mitotic body in the sclerotome associated with a non-muscle cell. Note that some nuclei have three nucleoli (tn). Scale bar, 25 µm.

transient epithelial spheres (Fig. 3A) that subsequently differentiated into mesenchymal derivatives. The somites were difficult to count accurately with a binocular microscope until the following day. Mitotic bodies were relatively common in the cells of the epithelial somites (arrows in Fig. 3A). New somites were added at the rate of approximately one every 6 h until the full complement of 62–63 somites was formed approximately 35 days post-fertilization (Fig. 4). ANCOVA of six families of NSR salmon revealed significant family differences in the rate of somite formation (F5,296=2.27; P=0.047), but no effects of ploidy. An examination of serial sagittal sections indicated that the first myotubes formed adjacent to the notochord at the level of the horizontal septum (long arrows in Fig. 3B). Multinucleated myotubes formed in a gradient away from the horizontal septum. Myoblasts apparently exited the cell cycle following fusion since mitotic bodies were never observed within myotubes (Fig. 5A,B). A wave of myotube formation

Salmon muscle growth 1997 70

Fig. 4. Somite formation at 6 °C in full-sibling normal-sex-ratio diploid (open symbols) and triploid (filled symbols) families of Atlantic salmon. Only the data from families 1 and 5 are shown for clarity. Second-order linear regressions were fitted to the data. Family 1 (circles): for diploids (line omitted for clarity), N=−89.22+6.40t−0.049t2 (r2 adjusted=0.96; P=