Muriel Mambrini

Voluntary feed intake, nitrogen and phosphorus losses in rainbow trout (Oncorhynchus mykiss) fed increasing dietary levels of soy protein concentrate

High energy extruded diets were formulated to contain the same level of protein supplied either by soy protein concentrate (SPC) or fish meal. Three experiments were performed in order to measure voluntary feed intake and feed waste, faecal losses and soluble losses of nitrogen and phosphorus in rainbow trout (average body weight: 100 g). Voluntary feed intake and growth performance of fish fed with demand feeders were not different when diets contained 0, 50 or 75 % SPC instead of fish meal. Total replacement of fish meal by SPC led to a significant decrease in feed intake and resulted in poor growth. This was partly due to methionine deficiency in the SPC based diet. With the addition of crystalline DL-methionine in the diets, an improvement of feed intake and growth performance was apparent. Protein digestibility was high, regardless of the protein source. Excretion of ammonia and urea increased with the level of SPC in the diet. Nitrogen losses decreased when methionine was added to the diet containing only SPC as a protein source. Availability of phosphorus increased with the level of SPC in the diets. Daily soluble losses were not affected by the dietary treatments but the pattern of phosphorus excretion after feed intake was modified. The rise in soluble phosphorus in water occurred later when fish were fed diets with soy protein whatever the dietary level of soy protein concentrate.

The optimum dietary indispensable amino acid pattern for growing Atlantic salmon ( Salmo salar L.) fry

To determine the optimum indispensable (I) amino acid (AA) balance in Atlantic salmon (Salmo salar L.) fry, a single protocol established for the pig was adapted. The balance was calculated from the reduction in N gain after replacing about 45% of a single IAA by a mixture of dispensable AA in isonitrogenous diets. We confirmed that the mixture of AA simulating the AA pattern of cod-meal protein and gelatine (46:3, w/w) was used with the same efficiency as cod-meal protein and gelatine. From the deletion experiment an optimum balance between the IAA was derived. Expressed relative to lysine = 100, the optimal balance was: arginine 76 (SE 0.2), histidine 28 (SE 2.2), methionine + cystine 64 (SE 1.7), phenylalanine + tyrosine 105 (SE 1.6), threonine 51 (SE 2.4), tryptophan 14 (SE 0.7), valine 59 (SE 1.7). No estimates were made for isoleucine and leucine. Expressed as g/16 g N, the optimal balance was: arginine 4.0 (SE 0.0), histidine 1.5 (SE 0.1), lysine 5.3 (SE 0.2), methionine + cystine 3.4 (SE 0.1), phenylaline + tyrosine 5.6 (SE 0.1), threonine 2.7 (SE 0.1), tryptophan 0.7 (SE 0.0), valine 3.1 (SE 0.1). This AA composition is close to that of the Atlantic salmon whole-body, but using it as an estimation of the IAA requirements may lead to an overestimation of the branched-chain AA requirements and an underestimation of aromatic and S-containing AA requirements. The results are discussed in accordance with the key assumptions associated with the model used (broken-line model, IAA efficiencies and maintenance requirements).

Partial replacement of dietary protein nitrogen with dispensable amino acids in diets of Nile tilapia, Oreochromis niloticus

Eighteen groups of 40 juvenile tilapia (Oreochromis niloticus) were fed five experimental diets in which crude protein (30% dry matter) was replaced at a level of 25% by dispensable amino acids (DAA) in the free form, given singly (alanine, glutamic acid or glycine) or as a mixture and at a level of 50% by DAA mixture. At the 25% substitution level, irrespective of the nature of the DAA, growth was slightly reduced (10%), while glycaemia, nitrogen retention and excretion were unaffected. A 50% substitution with the DAA mixture resulted in a marked reduction in growth (50%), a modified pattern of glycaemia and a lower nitrogen retention associated with a higher nitrogen excretion. Results suggest that DAA should not account for more than 60% of the total amino acid supply in tilapia diets.

Enhanced individual selection for selecting fast growing fish: the "PROSPER" method, with application on brown trout (Salmo trutta fario)

Growth rate is the main breeding goal of fish breeders, but individual selection has often shown poor responses in fish species. The PROSPER method was developed to overcome possible factors that may contribute to this low success, using (1) a variable base population and high number of breeders (Ne > 100), (2) selection within groups with low non-genetic effects and (3) repeated growth challenges. Using calculations, we show that individual selection within groups, with appropriate management of maternal effects, can be superior to mass selection as soon as the maternal effect ratio exceeds 0.15, when heritability is 0.25. Practically, brown trout were selected on length at the age of one year with the PROSPER method. The genetic gain was evaluated against an unselected control line. After four generations, the mean response per generation in length at one year was 6.2% of the control mean, while the mean correlated response in weight was 21.5% of the control mean per generation. At the 4th generation, selected fish also appeared to be leaner than control fish when compared at the same size, and the response on weight was maximal (≈130% of the control mean) between 386 and 470 days post fertilisation. This high response is promising, however, the key points of the method have to be investigated in more detail.

Genetic variability in residual feed intake in rainbow trout clones and testing of indirect selection criteria (Open Access publication).

Little is known about the genetic basis of residual feed intake (RFI) variation in fish, since this trait is highly sensitive to environmental influences, and feed intake of individuals is difficult to measure accurately. The purpose of this work was (i) to assess the genetic variability of RFI estimated by an X-ray technique and (ii) to develop predictive criteria for RFI. Two predictive criteria were tested: loss of body weight during feed deprivation and compensatory growth during re-feeding. Ten heterozygous rainbow trout clones were used. Individual intake and body weight were measured three times at threeweek intervals. Then, individual body weight was recorded after two cycles of a three-week feed deprivation followed by a three-week re-feeding. The ratio of the genetic variance to the phenotypic variance was found high to moderate for growth, feed intake, and RFI (VG/VP = 0.63 ± 0.11, 0.29 ± 0.11, 0.29 ± 0.09, respectively). The index that integrates performances achieved during deprivation and re-feeding periods explained 59% of RFI variations. These results provide a basis for further studies on the origin of RFI differences and show that indirect criteria are good candidates for future selective breeding programs.

Stable and full rescue of the pigmentation in a medaka albino mutant by transfer of a 17 kb genomic clone containing the medaka tyrosinase gene.

In the medaka Oryzias latipes, several albino strains have mutations in the tyrosinase gene that have been fully characterized at the molecular level. A genomic clone from wild-type medaka containing the 5 kb tyrosinase gene with its five exons, 10 kb of upstream sequences and 2 kb downstream sequences was introduced into fertilized eggs from a tyrosinase-negative albino strain. We show that the injection of this genomic clone predominantly conferred mosaic expression ending before the hatching stage. A minority of juveniles retained a variable number of pigmented cells, including four individuals keeping one pigmented eye through adulthood. Two of these could be mated, and one of these transmitted the transgene resulting in complete rescue of pigmentation to 16% of its offspring. The resulting transgenic line harbors a single copy of the wild-type tyrosinase gene and all fish are wild-type with respect to pigmentation. These experiments suggest that the tyrosinase genomic clone, or a future shorter version of it, can be used in fish to routinely detect transgenic lines. The apparent faithful and systematic expression of the tyrosinase transgene is most probably due to the presence of a locus control region (LCR) in the injected clone.

Analysis of Cell-Specificity and Variegation of Transgene Expression Driven by Salmon Prolactin Promoter in Stable Lines of Transgenic Rainbow Trout

In order to identify the specificity and functionality of salmon prolactin (sPRL) promoter, transgenic rainbow trout carrying a construct comprising the 2.4 kb fragment of the 5′ flanking region of Atlantic Chinook sPRL gene fused either to the reporter genes cat (sPRL-cat) or lacZ (sPRL-lacZ) were produced. sPRL-cat in transgenic F0 fish expressed strongly CAT only in the pituitary gland. Transgenic in F1–F4 lines harbouring sPRL-lacZ expressed β-galactosidase (β-gal) only in the follicular PRL-producing cells of the adenohypophysis. We observed heterocellular, mosaic distribution of β-gal within PRL cell population and enormous variation of lacZ expression level between the littermates in the same transgenic line. Regardless of the transgene copy number, age or sex of transgenic fish, β-gal expression was lactotroph-specific but variegated in all the nine F2 hemizygous lines analysed. One line harbouring a multicopy integration was followed up to F4 generation: the transgene was transmitted without modifications. Analysis of genomic DNA from pituitaries showed that lacZ sequences were highly methylated. LacZ expression was low and its transcripts, analysed by in situ hybridisation, showed a mosaic distribution within the pituitary gland. These data suggest that variegated expression of lacZ can occur at the transcription level owing to the silencing effect of lacZ gene. After proving the tissue-specific expression of reporter genes driven by the sPRL promoter, we tried to obtain the genetic ablation of PRL-producing cells, by transferring the same construct comprising diphtheria toxin DT-A gene (tox). However, the high mortality rate of sPRL-tox transformed embryos has embedded this study and no transgenic fish expressing tox were produced. The appropriateness of using transgenic strategies to analyse gene function in Salmonids is discussed, especially the implications of the multicopy integration patterns and of the variegated transgene expression.