Background of the Invention

1. Technical Field

The present invention relates to methods for obtaining improvements to the body composition of fish. More particularly, the present invention provides methods for increasing lean (protein) mass, increasing moisture content and decreasing body fat mass of fish via the administration of porcine somatotropin.

2. Background Art Numerous studies have demonstrated the efficacy of various somatotropins for generally enhancing weight gain in fish. Recombinant bovine, human, as well as a bovine analog of somatotropin have been shown to be effective in inducing weight gain in various fish, such as trout, salmon, and catfish. Down et al . , in Growth Acceleration of Seawater-Adapted Female Chinook Salmon . . ., J. World Aqua. Soc. 20:4, 181-187 (1989) demonstrated that recombinant bovine growth hormone (rbGH), administered in a continuous fashion by osmotic pumps, can accelerate the growth of sub-adult chinook salmon. Growth was measured as increases in length and weight only - no body composition analyses were reported.

In an earlier article, Down et al. showed that rbGh and an analog of rbGH ( "21K-rbGH" ) caused increases in weight and length, but no change in moisture content, of coho salmon to which the hormones were administered. Changes in body composition (that is, percent fat or lean meat mass) were not reported. Down et al. did observe, however, that the "condition factor" of the treated fish (condition factor is a function of weight ÷ length) was actually reduced in comparison to untreated fish over a portion of the treatment period. The authors attributed this observation to effects of somatotropin on bone growth. See Down et al. , A Potent Analog of Recombinant Bovine Somatotropin . . ., Can. J. Fish. Aquat. Sci. 46, 178- 183 (1989).

R. Wilson et al., in Effect of Recombinant Bovine Growth Hormone Administration on Growth and Body Composition of Channel Catfish, Aquaculture 73, 229- 236 (1988), report that the administration of rbGH to fingerling channel catfish results in an increase in weight gain, but that the increase is due to increased fat deposition. Body composition analysis showed both an increase in fat deposition and a decrease in body protein content of fish receiving rbGH when compared to the controls.

Skyrud et al., Effects of Recombinant Human Growth Hormone and Insulin-like Growth Factor 1 on Body Growth and Blood Metabolites in Brook Trout ( Salvelinus fontinalis), Gen. and Comp. Endocrin. 75, 247-255 (1989), report that the administration of human growth hormone resulted in overall increased growth (weight, length and liver weight) of trout. Human IGF-1, another hormone known to stimulate growth in mammals, had no growth-stimulating effect on trout, however. In

higher doses, IGF-1 actually retarded growth and increased mortality.

In Komourdijan et al. , The Effect of Porcine Somatotropin on Growth, and Survival in Seawater of Atlantic Salmon ( Salmo salar) Parr, Can. J. Zool. 54, 531-535 (1976), the authors report that the administration of porcine somatotropin results in increased length and weight, and decreased condition factor, over non-treated controls. Again, no body composition data were presented by the authors.

Porcine somatotropin (pST) is a well-characterized polypeptide hormone synthesized in and secreted from the anterior pituitary. pST derived from porcine pituitary gland is available from commercial sources (Sigma Chemical Co., St. Louis, Mo., U.S.A.).

Recombinant pST and various bio-active fragments and analogs thereof also have been described in the literature. A particularly preferred pST-like polypeptide, pST-Δ7, is described in published European Patent Application No. 104,920 Al (Biogen N.V., published April 4, 1984). This same polypeptide is referred to as Δ7pGH in Biogen's U.S. patent no. 4,766,224, the disclosure of which is incorporated by reference herein. From the foregoing, it is apparent that, prior to the present invention, no clear conclusions could be drawn regarding the effects of any particular heterologous somatotropin on fish. While some researchers reported overall weight and length gains, as least one group (Wilson et al.) reported increased body fat and decreased protein mass in treated fish. Thus, improvements in fish propagation techniques, and, specifically, in lean mass enhancement and fat reduction, remain a goal of the fisheries industry.

Accordingly, one of the objects of the present invention is to provide a method for enhancing the growth of fish and, specifically, for increasing the lean (protein) mass and decreasing the body fat content.

Summary of the Invention

The foregoing and other objects are achieved by the present invention which, in one aspect, provides a method for increasing the lean meat mass and decreasing the body fat mass of fish. The method comprises administering to a fish an amount of porcine somatotropin effective to achieve the same. The porcine somatotropin preferably is administered parenterally. In the most preferred aspects of the invention, the recombinant pST-like polypeptide pST-Δ7 is administered. The administration of this polypeptide, as described in detail below, is seen to provide significant increases in lean mass, and significant decreases in body fat composition, when administered to fish.

Brief Description of the Drawing

Figure 1 is a plot of trout body weight gain versus time for a study conducted according to the present invention.

Detailed Description of the Preferred Embodiments

In accordance with the present invention, porcine somatotropin is administered on a periodic basis to fish. Advantageously, overall body weight gains and improvements in feed efficiency are attained. More importantly, however, it has now been discovered that the administration of pST to fish provides marked

improvements in body composition as well. The improved body composition attributes include increased lean mass, decreased fat mass and increased moisture content. As mentioned briefly above, pST is available both as a natural product, derived from porcine tissues, as well as a recombinant product. In view of the substantial costs associated with obtaining pST from animal tissues, recombinantly produced pST and its structurally related derivatives and analogs are preferred.

A particularly preferred recombinant, pST-like polypeptide is pST-Δ7. This polypeptide has an amino acid sequence similar to that of native pST, but is missing the first 7 amino acid residues and includes an f-Met residue. pST-Δ7 and recombinant vectors for its expression in, for example, E. coli are described in EP 104,920 which was mentioned earlier and is incorporated by reference herein. The pST may be administered to the fish in a variety of manners, including orally, parenterally, via an implanted dosage form or via an implanted osmotic pump. When orally administered, pST can be incorporated into a fish food preparation. pST for injection will be mixed with any of the known pharmaceutical carriers or vehicles which are suitable for use in the administration of pharmaceutical preparations to fish. Similarly, implantable dosage forms of somatotropins, including pST, are well known. In general, the amount of pST which is administered to the fish is defined as an amount effective to bring about the desired body composition changes - increased lean (protein) mass, decreased body fat mass and increased moisture content. At dosages

sufficient to bring about these effects, other beneficial effects such as generally increased body weight and size, and enhanced feed efficiency, will be realized as well. Advantageously the protein content of the fish will be increased by at least about 1% (by weight) over a 12-week growth period, while the fat content of the fish will be decreased by at least about 1% over the same period.

One mode of administration of pST in accordance with the present invention is based on periodic administration via parenteral injection. A pST dosage consisting of, for example, about 1 to about 15 μg per gram of body weight, preferably from about 5 to about 10 μg/g, administered parenterally at 7 or 14 days intervals, has been found to provide significant (p<0.05) alterations to moisture, fat and protein content. Other modes of administration will be apparent to the ordinarily skilled scientist.

In one series of experiments which demonstrate the efficacy of the invention, a total of 400 rainbow trout ( Oncorhynchus mykiεε) within one standard deviation of the initial population weight were randomly selected and equally distributed to 20 constantly aerated tanks (225 cm long X 42.5 cm deep X 25 cm wide). Water mean temperature was maintained with spring water at 15°C. A 12 hour light - 12 hour dark photoperiod was maintained. The trout were fed Rangen Salmon Grower pellets (Table 1) except 24 h before handling. Feed offered was increased on a daily basis to insure that feed was not growth limiting. A 3/32" pellet was fed for the first two weeks followed by a 50:50 mixture of the 3/32 and 1/8" pellet for a week, then the 1/8" pellet for the remainder of the trial. The fish were

allowed two weeks to adapt to the environment before initiation of the study.

Table 1 Composition of Rangen Salmon Grower Feed Component (%) Pellet (1/8") Pellet (3/32")

Protein 50.4 48.9

Fat 15.7 16.3

Moisture 8.02 8.49

Ash 9.91 8.92 Crude fiber 0.73 1.26

The treatments consisted of pST (Δ7pGH as described in U.S. Patent No. 4,766,224) administered at 14-day intervals at doses of 0, 5 or 10 μg/g body weight and at 7-day intervals at doses of 5 μg/g body weight. Lyophilized pST was dissolved in an aqueous diluent (0.2 M tris, 2mM EDTA and 0.15% BRIJ-3) to which was added streptomycin sulfate (lOOμg/ml), Penicillin G (100 IU/ml), and amphotericin B (0.25 μg/ml) for prophylaxis. Administration volume was 1 μl/g body weight.

Trout were netted from the troughs, anesthetized in methane tricaine sulfonate (100 ppm), blotted with moist absorbent towels, weighted to the nearest 0.1 g, and injected to the nearest μl into the body cavity along the ventral body wall behind either pectoral fin with Hamilton syringes and 27-gauge needles. Immediately after the injection, the trout were placed in an aerated recovery bath containing 5 ppt NaCl and 5 ppm Chloramine-T for disease prophylaxis. After recovery, the fish were returned to the appropriate trough.

Ten fish per tank were selected for body composition analysis at the start of the study. At the end of the 12-week study, five randomly selected fish per tank were selected for body composition analysis. Feed was withheld from these fish for 48 hours before sacrifice. Whole carcasses were ground and sub-sampled for analysis. The samples were analyzed for fat by ether extraction, for total moisture content by oven drying at 100°C and for lean mass (protein) content by Kjedahl analysis (AOAC, 1980).

The study was conducted as a randomized block design. The data collected and/or calculated included initial weight, final weight, average daily gain, feed conversion efficiency, chemical analysis of body composition and protein and fat accretion rates. The data were analyzed by analysis of variance with planned mean comparisons (SAS ^® , 1989).

Trout performance during the growth period is presented in Table 2 with weight gain illustrated in Figure 1. Initial trout weight was similar among the treatment groups. Final weight and average daily gain were significantly (p<0.01) greater with pST administration as compared to the control groups. Administration of 5 or 10 μg/g body weight at 14-day intervals resulted in a 15% and 8% improvement in average daily gain, respectively, as compared to the control. A 41% improvement in average daily gain was observed when 5 μg/g pST were administered on a weekly basis, as compared to the control. Even though there was no significant difference between the performance of the two control groups, performance appears poorer with the fish being handled weekly as compared to biweekly. This probably results from the increase in stress due to more frequent handling.

Feed conversion efficiency was significantly (P<0.01) improved in trout treated with pST as compared to the controls. Administration of pST on a biweekly basis resulted in an 11% and 8% improvement in feed efficiency for the 5 and 10 μg/g treatments, respectively, as compared to the control. When pST was administered on a weekly basis, a 20% improvement in feed efficiency gain was observed as compared to the control. This level of improvement is considerably greater than that previously reported for other somatotropins and types of fish.

Whole body composition as well as fat and protein accretion rates of the trout at the completion of the trial are presented in Table 3. Administration of pST to trout significantly (P<0.05) altered the body content of moisture, fat and protein. Both moisture and protein were increased 5% for both the 5 and 10 μg/g biweekly treatments as compared to the controls. However, the 5μg/g weekly treatment only increased protein content 1% as compared to the control. Protein accretion was significantly (P<.01) enhanced by pST administration as compared to the controls. Protein accretion was increased by 21 and 13% for the fish treated biweekly with 5 or 10 μg/g, respectively, as compared to the controls. Treatment of trout with 5 μg/g weekly resulted in a 42% increase in protein accretion as compared to control fish. The fat content was decreased 7 and 11% for the 5 and 10 μg/g biweekly treated fish, respectively, as compared to the controls. When the pST was administered at 5μg/g weekly, a 13% reduction in fat occurred as compared to the controls. Fat accretion was not significantly (p>0.10) affected by treatment. This is believed to be the first demonstration of enhanced body protein

accretion and decreased percent body fat content of fish as a result of somatotropin administration.

Table 2

Effect of pST Administration on the Growth Performance of Rainbow Trout

0 pST/14 vs 0 pST/7 NS No pST vs pST NS

0 pST/7 vs 5 pST/7 NS

0 pST/14 vs 5 pST/14 NS

Table 3

Effect of pST Administration on the Whole Bod Com osition of Rainbow Trout

CV 1.4 9.1 1.8 11.6 15.2

Contrasts P values

0pST/14 vs 0 pST/7 NS NS .0319 .0009 NS No pST vs pST .0055 .0150 .0168 NS NS 0 pST/7 vs 5 pST/7 .0452 .0449 NS ,0384 NS 0pST/14 vs 5 pST/14 NS NS .0110 ,0012 NS

Although the present invention has been described in connection with certain preferred embodiments and specific examples, it is not so limited. Variations with the scope of the claims will be readily apparent.