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ANIMAL GENETICS |
* INRA, Laboratoire de Génétique des Poissons, 78350 Jouy-en-Josas, France;
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Unité Nutrition, Aquaculture et Génomique, 64310 Saint Pée sur Nivelle, France; and
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SEMII, 29450 Sizun, France
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionImplicationsLiterature Cited |
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Key Words: Correlated Responses • Energy Requirements • Feed Intake • Growth Rate • Salmo trutta • Selection
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionImplicationsLiterature Cited |
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; Vandeputte et al., 2002). Although selection for growth is more recent than in other farmed animals, the selected lines of fish are now sufficiently divergent to carry out an analysis of the correlated responses. As in higher vertebrates (Rauw et al., 1998), the enhanced growth rates are explained by an increased feed intake associated (Thodesen et al., 1999) or not associated (Sanchez et al., 2001) with improved G:F. Response variability may be explained by the different selection procedures (familial or individual), the selection criteria (BW or length), the basis of comparisons (wild or domesticated control lines), and the ways fish are fed and feed intake is measured. In fish, the group level is generally the experimental unit. This implies specific experimental designs, particularly when intake is considered.
Brown trout have been selected by an enhanced individual procedure since 1987 (Chevassus et al., 1992), and a control line has been maintained. It seems that selected fish exhibit their growth potential when fed ad libitum only (Sanchez et al., 2001), and G:F may not be affected by selection. This suggests that the nutritional requirements are not different between the lines. The objective of this study was to verify that G:F is not affected by selection. Our strategy was to test the robustness of the response by analyzing the genotype x environment interactions when the lines are reared under conditions known to affect feed efficiency: feed restriction (Cho, 1992) and conditions for compensatory growth (Dobson and Holmes, 1984). The specific effects of such rearing conditions are not well known in brown trout, so they will be described first in the controls. The specific effect of selection will then be assessed. In addition, the experiment was designed to compare maintenance and growth requirements of both lines. Indeed, the specific effect of selection for growth on nutritional requirements has never been described in fish.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionImplicationsLiterature Cited |
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). From the same initial population, a control line (C) was maintained under the same rearing conditions. With a selection pressure on L of approximately 5% (proportion of fish selected), the correlated weight gain at 1 yr of age was approximately 24% per generation (on average over four generations) for the selected line (S) compared with the C (Vandeputte et al., 2002). The S and C groups of the current study were produced by in vitro fertilization of 34 S females crossed with 11 S males and 27 C females crossed with 37 C males. To obtain selected and control offspring reaching a similar weight of approximately 4 g at the beginning of the experiment, we took into account the value of the growth rate usually observed for such lines (our unpublished data; Sanchez et al., 2001) and calculated that C had to be fertilized 12 d before S. After hatching, fingerlings were reared in two tanks per line supplied with 11° C flow-through water. They were fed ad libitum a commercial dry feed (Biomar Ecolife 18 (Nersac, France) containing 51% CP and 18% lipid (data from the manufacturer) by automatic feeders delivering food 12 h/d until the beginning of the experiment. The day before allotment, the distribution of length of the two populations was analyzed to optimize the constitution of the experimental groups. This was based on individual L of 580 C (range recorded = 55 to 81 mm) and 782 S (range recorded = 55 to 91 mm) fish. The individual BW were also recorded. Trout were then sorted to obtain homogeneous groups with L between 60 and 80 mm. The effect of the sorting was assessed after controlling the distribution of BW and L in the sorted populations (299 C and 302 S). Each line was divided into nine sets of 100 and nine sets of 150 fish reared in 180 L flow-through tanks supplied with bore hole water (temperature 11 to 12° C).
The experiment was conducted in a manner compatible with national legislation on animal care. In application of the French penal and rural code, M. Mambrini and T. Boujard have personal authorization delivered by the French Agricultural Ministry for conducting animal experiments (Authorizations 5625 and 5635) and ensured that all procedures used in this experiment were ethically acceptable and followed the procedures stipulated by the French Ministry of Research.
Experiment
The experiment was divided into three successive phases: 1) the feeding phase, where fish were fed six different feeding levels; 2) the feed deprivation phase; and 3) the refeeding phase, where all groups of fish were fed in excess.
During the feeding phase, groups of 150 fish were fed at 10, 30, or 50%, whereas groups of 100 fish were fed at 70, 100, or 140% of the usual daily ration (UDR, calculated using the prediction software "Ecureuil," developed at the SEMII fish farm, taking into account the fish species, the water temperature and the weight of the fish). The group size was chosen so that the biomass expected at the end of the experiment remained much lower than 100 kg/m3, a biomass under which growth performance remains optimal in salmonids (Boujard et al., 2002). The number of individuals in the more feed restricted groups was higher compared with the other groups because of the expected mortality. Moreover, the induced difference in density does not affect the behavior of the fish at this biomass level (Boujard et al., 2002). The fish were fed by automatic feeders in a 2 x 6 x 3 factorial design (line x feeding level x replicates). The uneaten food was collected at the outlet of each tank three times per week, dried, and weighed, and this weight was subtracted from the amount of food distributed to calculate the actual amount of feed consumed. Fish were fed for 116 d, except those fed 10 and 30% UDR, for which the experiment was stopped after 82 d. As a higher mortality was expected in those groups, plans were implemented to stop a feeding level if mortality was close to 10% of the assigned individuals. Only the fish fed 50 to 140% UDR were included in the next two phases of the experiment.
The phase of feed deprivation lasted for 35 d. During the refeeding phase, fish were all fed 140% UDR for 47 d. As during the feeding phase, fish were fed by automatic feeders and the actual amount of feed consumed was deducted, taking into account the uneaten food.
The diet used throughout the experiment contained (DM basis) 49.4% CP, 25% crude fat, and 23.7 kJ of GE/g. This high-protein, high-energy diet consisted of extruded pellets of different diameters that were kept at 4°C until use. Pellet size was chosen according to the fish size.
Measurements and Calculations
Mortality was recorded daily throughout the experiment and the weight of the dead fish was taken into account for weight gain calculations. At the end of each experimental phase (feeding, feed deprived, and refeeding) for the groups initially fed 50 to 140% of their UDR, and on d 82 only for those fed 10 and 30% UDR, the fish were counted and weighed by group after 1 d of fasting. Growth coefficients were calculated according to Cho (1992), using the daily growth coefficient (DGC), a value independent of the BW. Gain:feed ratios were calculated taking into account the actual feed consumed. Individual weights (grams, noneviscerated) and fork lengths (to the nearest millimeter) were recorded for all fish. Coefficients of variation of weight, length, and the conformation coefficient (K-factor) were calculated within each tank.
Twenty fish were initially sampled for whole body composition. The whole body samples were frozen (–20°C), ground, and freeze dried. At the end of the feeding and feed deprivation phases, 10 fish per tank were sampled and immediately frozen for subsequent analysis. The following analyses were performed on the diet and whole carcasses: DM, CP (N x 6.25), GE, and fat (AOAC, 1990). The water content was calculated by weight loss after drying at 110°C for 24 h in a forced-air oven. Nitrogen analysis of all samples was performed by the Kjeldahl method. The GE content of the diet was determined with an adiabatic bomb calorimeter (IKA, Staufen, Germany). Fat content was calculated after extraction in a Soxhlet apparatus using petroleum ether as solvent.
Protein and energy gains were plotted against protein and energy intake expressed as g/(kg BW0.75•d) and kJ/ (kg BW0.75•d), respectively, to estimate maintenance and growth requirements. Data were adjusted using linear model and saturation kinetics model (Mercer, 1982), which both led to similar estimates. Maintenance requirements were interpolated using the linear model, as the amount of protein and energy intake resulting in protein and energy gain equal to zero (x-intercept), and requirements for growth were estimated as the inverse of the slope. In addition, for feeding levels higher than 30% UDR, protein and energy use for body accretion were calculated as the intake of protein and energy per kilogram of wet weight gain during the feeding phase. Nutrient losses during the food deprivation phase were deducted from the body composition data.
Statistical Analyses
Statistical analyses were based on a completely random experimental design. Group data were compared using an analysis of covariance, including the effects of line, feeding level, and interaction between these two factors, which were tested using the tanks as the experimental unit (three replicates per treatment). Percentage data were transformed (arcsine square root) before being subjected to the analysis.
For the group growth data, the covariate included in the model was the initial BW of each experimental phase. For the feeding phase, the initial BW was taken into account because of a slight difference between the two groups (see results). For the feed deprivation and the refeeding phases, the mean BW at the end of the previous phases were used as a covariate. For the body composition data, the covariate included in the model was the final BW because it is accepted that body composition greatly influences BW (Shearer, 1994).
Individual data were compared using an ANOVA including the effects of the line, the feeding level, the interaction between these two factors, and the effect of the tank nested into this interaction (100 to 150 individuals per tank, with three replicates per treatment).
Probabilities of differences between treatments and residual standard deviation were generated after ANOVA was performed using the GLM procedure of SAS (SAS Inst., Inc., Cary, NC). When the interaction was significant, the respective effects of the line and the feeding level were tested separately with one-factor ANOVA. The means were subsequently compared using the test of Newman and Keuls (with P < 0.05).
To compare the extent of the absolute variation for each individual variable (BW, length, K-factor), they were transformed to logarithms, and the equality of the absolute deviates in each class was controlled. The absolute deviate was calculated as (|ln Yij – ln 
j|), where Yijis the jth term in the ith sample, and lni is the mean logarithm of the ith sample. The scatter of the absolute deviates was markedly unequal, indicating that a nonparametric test has to be applied (Sokal and Braumann, 1980). The CV were thus compared with the nonparametric test of Kruskal-Wallis using the NPAR1WAY procedure of SAS. The line and feeding level effects were tested separately (three replicates per treatment).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionImplicationsLiterature Cited |
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Survival rate at the end of the experiment was higher than 95% in groups fed at least 50% UDR. For groups fed less than 50% UDR, the experiment lasted only for 82 d, and mortality ranged between 88 and 90%.
Feeding Phase
During the feeding phase, the effect of the line on daily growth coefficient and feed intake depended on feeding level (Tables 1 and 2). Daily growth coefficient was significantly different (P < 0.001) between lines only when they were fed 100 and 140% UDR. For groups fed 10 to 70% UDR, no uneaten feed was detected, so it was considered that all the feed distributed was eaten. At these feeding levels, cumulative feed intake was not different between C and S (Tables 1 and 2). At 100% UDR, uneaten feed was observed for C only (8.2 ± 2.3% of the distributed feed). At 140% UDR, the proportion of distributed feed that was uneaten was higher in C (14.9 ± 3.2%) than in S (9.4 ± 0.9%). Thus, the main differences between lines in fish fed 100 and 140% UDR was in their respective cumulative feed intake (Table 2). Gain:feed ratio was affected by the feeding level, being lower at the lowest feeding levels (10, 30, and 50% UDR) and maximal at 100% UDR. Whatever the feeding level, G:F was never different between lines.
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and 2).
The protein and energy gains increased linearly with the protein and energy intake, respectively (Figure 1), without any differences between lines. The estimated maintenance requirements were similar among the lines: 8.1 and 8.5 g of CP/(kg BW0.75•d) and 446 and 433 kJ of GE/(kg BW0.75•d) for S and C lines, respectively. The growth requirements were also comparable between the lines: 1.53 and 1.44 g of CP/g of protein gain and 1.37 and 1.35 kJ of GE/kJ of energy gain for S and C lines, respectively. The amounts of CP and GE utilized to produce 1 kg of wet weight gain were also similar for both lines at each feeding level tested (Table 2). The values were the lowest at 100% UDR, and the highest at 50 and 140% UDR for both lines.
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and 2). Regardless of the feeding level, S was always longer (P < 0.001) than C. Except for fish fed 140% UDR, the K-factor was always lower (P < 0.001) in S than in C. The apparent discrepancy at 140% UDR could be attributed to the large difference (P < 0.001) in feed intake and final BW between the C and S.
Variability of the final BW and L was higher in fish fed 30% than in those fed 10% UDR (Table 1). For higher feeding levels (Table 2), the variability decreased with the level of feed intake. In addition, at the highest feeding levels (100 and 140% UDR), BW and L variability were lower in S than in C. Because maximal intake was higher for S than for C, the CV in weight were plotted against the observed level of feed restriction (cumulative intake at a given feeding level/cumulative intake measured at 140% UDR; Figure 2). This shows that the variability in the final weight of S is always lower than that of C at the same restriction level.
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), the relative loss of BW was not significantly affected (P > 0.07) by either the line or the previous feeding level (P > 0.18). The relative daily loss of BW was only slightly higher in S than in C for a previous feeding level of 140% UDR. Body composition was not affected by the previous feeding level or by the line. Relative losses of nutrients (protein loss, –0.97 g•kg–1•d–1 for C and S, P > 0.57; lipid loss, –0.58 and –0.67 g•kg–1•d–1 for C and S, respectively, P > 0.22; energy loss, –53 and –55 kJ•kg–1•d–1 for C and S, respectively, P > 0.38) also were unaffected by feeding level and line. The K factor values were much lower at the end of the feed deprivation phase than at the end of the feeding phase. The line and the previous feeding level affected body conformation in a way similar to that at the end of the first experimental period. This indicates that there was no interaction between the effect of 35 d of feed deprivation and the line or the previous feeding level. Briefly, S fish remained slightly leaner than C fish when previously fed restricted rations, whereas they were fatter than C when previously fed 140% UDR. The tendency for a higher heterogeneity in BW (P < 0.001) and L (P < 0.007) in C than in S remained after this period of feed deprivation.
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), fish were fed 140% UDR, and uneaten feed was recovered in each tank. This indicates that all the groups were fed in excess; thus, the cumulative feed intake reflects maximal voluntary intake. Growth performance was high in all groups and significantly better for S than for C. This was largely explained by the higher cumulative intake (P < 0.001) of S compared with C. The lower the previous feeding level, the higher the growth rate during the refeeding period. There was no clear effect of the line on G:F. At the end of the refeeding period, S was still longer than C (P < 0.001). The K-factor was much higher compared with the values observed at the end of the feed deprivation phase, but no clear differences appeared between the lines. With the exception of the fish previously fed 50% UDR, the CV in BW and L were lower for S than for C.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionImplicationsLiterature Cited |
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), maximal feed intake of brown trout is tightly regulated. A slight decrease in G:F was seen at the 140% UDR (i.e., in overfed fish). This negative effect of a high feeding level on G:F has already been observed in other fish species (Storebakken and Austreng, 1987a,b; Ballestrazzi et al., 1998; Valente et al., 2001). It has been assumed to result from increased energy expenditure, namely a higher heat increment of feeding to sustain protein and fat deposition (Storebakken et al., 1991). Another explanation is that it is difficult to assess with accuracy the amount of uneaten feed in water running out of the tank when feed is distributed in excess. As discussed further, the amounts of uneaten feed were much lower for selected fish fed 100 and 140% UDR than for the controls, whereas the G:F were similar among the two lines, which is in favor of a negative effect of high feeding levels on feed efficiency. Indeed, dietary protein and energy use efficiencies were negatively affected when fish were fed in excess.
A negative relationship between feed intake and BW heterogeneity was observed. It is well known that in salmonids, when the access to food is qualitatively or quantitatively restricted at the group level, the interin-dividual variability of feed intake, and thus variability in individual growth performance, is increased (McCarthy et al., 1992; Jobling and Koskela, 1996; Gélineau et al., 1998; Kristiansen, 1999). The observed weight heterogeneity in feed-restricted groups may be explained by individual intake heterogeneity. Other results obtained in the current study support this assumption. At the end of the feed deprivation phase, BW and length variabilities were similar to those observed at the end of the feeding period. When the fish were refed, the heterogeneity of the groups previously restricted was largely reduced, whereas it remained similar for the groups previously unrestricted. One might argue that the variability in individual intake is linked, at least in salmonids, to increased competition for food when fish are restricted (Davis and Olla, 1987). However, the share of food may depend on the strength of social interactions. They seem to play a larger role in brown trout than in rainbow trout (Kristiansen, 1999).
Increased feed intake led to a carcass fat concentration increase, and this is also reflected by a K-factor increase. At the end of the feed-deprivation phase, the K-factor was largely diminished and fish have lost about the same relative BW (approximately 16%), regardless of the previous feeding level. In addition, there was no effect of the previous feeding level on the extent of lipid and energy losses. This is in line with the fact that in our experiment, there was no link between the carcass fat concentration at the beginning of the feed-deprivation phase and the extent of weight loss. These results are surprising because during fasting, brown trout are known to mobilize in priority their perivisceral fat (Navarro et al., 1992). One might suggest that in the present experiment, the length of the feed deprivation phase was too short to observe any effect of fattening on weight loss. Indeed, brown trout are still able to mobilize the perivisceral fat after 50 d of feed deprivation (Navarro et al., 1992). These losses were in the same range as values reported for other salmonid species, as were the maintenance requirements (Cho and Bureau, 1995). Concerning the requirements for growth, we were unable to compare our results with published data because they were obtained on fish of different sizes and reared at different temperatures (Arzel et al., 1992). They seem lower than the recommendations given for rainbow trout (NRC, 1993). There are slight differences in the way brown trout and rainbow trout utilize nutrients for growth (Arzel et al., 1992, 1994). Our results indicate that brown trout may be more efficient than rainbow trout.
Growth rates exhibited during the refeeding phase were much higher than those observed during the feeding phase. Compensatory growth capacities have been demonstrated in several salmonid species (Dobson and Holmes, 1984; Jobling et al., 1994; Jobling and Koskela, 1996), and our results indicate that it is also the case for brown trout. In our experiment, as is usually observed, compensatory growth was achieved by a burst in both feed intake and G:F. In addition, the extent to which feed intake relative to BW increased was correlated to the strength of the feed restriction before the feed deprivation phase. Voluntary feed intake of fish may be affected by their past nutritional history, in particular the previous level of feed restriction (Boujard et al., 2000) and the duration of feed deprivation (Hayward et al., 1997). Taken together, these data show that in brown trout, feed intake is a key factor for the compensatory growth phenomenon.
Selected Fish
This study confirms that the main factor affected by our selection procedure for increased body length is feed intake. In selected lines, feed refusals were only observed at 140% UDR. At this feeding level, fish exhibited their best growth potential and reached a BW 55% higher than that of the control at the end of the feeding phase. This is in line with the values of the genetic gain expected at this age considering the average genetic gain obtained with this selection procedure (Sanchez et al., 2001; Vandeputte et al., 2002). This is also in agreement with the results obtained with other selection protocols for growth in salmonids (Gjedrem, 1998). At 100% UDR, selected fish were slightly restricted (no uneaten feed recovered, lower growth rates than when fed 140% UDR), showing that the level of voluntary feed intake was higher than in the control line. During the refeeding phase, the feed intake capacities of S were always higher than for C, and as observed in controls, intake relative to BW was all the higher as the previous restriction level was high, even though the feed intake capacities were always higher for S than for C. This high intake capacity appears to be a feature of the S line, remaining evident even after a period of feed restriction.
When feed intake was restricted, the S lines exhibited the same growth rate as the C line. The G:F remained comparable among the lines regardless of the feeding level, confirming on a larger scale our previous results (Sanchez et al., 2001). The main response correlated to this selection procedure was an increased intake capacity. In higher vertebrates, most of the heritable variation in BW is associated with differences in feed intake (Rauw et al., 1998). This also seems to be the case in fish taking into account estimates of heritabilities, which are not significantly different from zero for feed efficiency (Kinghorn, 1983) and range between 0.15 and 0.40 for feed intake (Kinghorn, 1983; Gjoen et al., 1991). Salmon selected with a familial procedure when compared with wild fish exhibit increases in both daily feed consumption and feed efficiency (Smith et al., 1988; Thodesen et al., 1999), but in these studies, the results merge the gains due to both genetics and domestication.
The lack of variability in feed efficiency is in line with the constancy of the body composition and nutrient retention among the lines. In addition, both lines seem to have the same needs for protein and energy gains, at least for the range of weights tested in the current study. Moreover, the maintenance needs were similar among the lines. This is also illustrated by the fact that selected and control fish lost the same relative weight and the same amount of protein, lipid, and energy during the feed-deprivation phase. Our selection procedure affected neither the nutrient requirements nor nutrient use efficiency. The increase in feed intake of the selected line in the current study is therefore disconnected from modifications of maintenance and growth requirements. In broilers, selection for increased BW was shown to damage the hypothalamic satiety mechanism, not to diminish hunger drive, and to lead to overeating (Burkhart et al., 1983; Denbow et al., 1986). Broilers are able to consume more than required until reaching the highest limit of their gastrointestinal capacity (Barbato et al., 1984) because of a higher rate of food gastrointestinal transit or because of increased digestion processes (Dunnington and Siegel, 1995). Such parameters could be specifically studied in our lines; however, their influence on feed intake is still largely unknown because in fish, the mechanisms governing feed intake regulation are marginally understood. Lines involved in this study might be good models for such investigations.
Intake in fish is measured on groups and, as exemplified in the current study, the food sharing and individual intake are affected by feeding level, whereas social interactions may influence individual intake. It may be that selection has affected the overall behavior. The interindividual variability of BW was lower in S than in C, regardless of the restriction level. This points to less competition for food in S fish. However, the decrease in the phenotypic variability of the selected trait has been observed in poultry after several generations of selection (Kawahara et al., 1974; Metodiev and Drbohlav, 1998). In our case, it does not seem to be linked to the genetic variability, which, based on enzymatic and microsatellite markers, was not affected by the selection procedure (B. Chevassus, unpublished results). Rather, the lower variability of individual BW of the selected line seems to be the result of decreased sensitivity to environmental factors. This may have positive implications in production because fish have to be re-sorted regularly due to their high interindividual BW heterogeneity.
The rearing management during selection may partly explain why selection for growth mainly increased intake capacities. During the selection process, fish were always fed in excess. Mice selected for growth under ad libitum feeding conditions exhibited increased intake capacities, whereas under feed restriction conditions, selection for growth decreased their appetite (Hetzel and Nicholas, 1978). The other important factor that may have influenced the results is that fish were selected based on length, and not weight. These two variables are closely linked, as indicated by the relative constancy of the K-factor in salmonids, at least when they are in the fast growing phase. Heavy but fatty fish might not have been retained during the selection procedure, which may explain the relative constancy of body composition. It has been demonstrated in pigs that a greater growth rate is achieved when animals are selected for lean growth efficiency (Chen et al., 2003; Fabian et al., 2003). Our results show that it may be difficult to improve metabolic efficiency with a selection for growth in fish. In higher vertebrates, the strategy was to select on residual feed intake (individual deviations from the regression of feed intake on BW gains). This selection procedure implies both that individual intake is accurately measured and that growth models have been correctly established (Kennedy et al., 1993). These conditions are still not fulfilled in fish. Individual feed intake cannot be measured in a continuous manner (Jobling et al., 2001). Growth models are only available for a few species. They are not commonly used and must integrate the constant improvements of the rearing performance observed in recently domesticated species. Then, a specific selection procedure is needed and indirect criteria, such as lean deposition, may be considered.
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Implications |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionImplicationsLiterature Cited |
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Footnotes |
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3 Present address: INRA, Station de Génétique Quantitative et Appliquée, Domaine de Vilvert, 78352 Jouy-en-Josas Cedex, France.
2 Correspondence: Domaine de Vilvert (phone: 33 1 34 65 27 05; fax: 01 34 65 23 90; e-mail: mambrini{at}jouy.inra.fr).
Received for publication December 17, 2003. Accepted for publication June 2, 2004.
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Literature Cited |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionImplicationsLiterature Cited |
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