IMPROVEMENTS IN OR RELATING TO FISH FARMING

Abstract: Fish are treated to produce an auto-immune response therein which impairs the development and/or maintenance of a normal gonad system in the fish. Such a response may be produced by the administration of antibodies to gonad tissue or of antigenic material derived from such tissue.

Description: IMPROVEMENTS IN OR RELATING TO FISH FARMING This invention relates to methods for modifying the sexual development of fish. Fish farming, particularly of salmonids, has developed rapidly throughout the world over the last ten years. However, one of the major unsolved problems associated therewith is that of the control of gonad maturation. Thus the growth rate of many fish falls off when sexual maturity is achieved and disease, particularly skin disease, becomes more prevalent. Moreover, it is preferred that male salmonids, for example, mature at the "salmon" (2 sea winter plus) stage of development when they are of a good size, but a proportion mature naturally as parr or as grilse. This can lead to increases in mortality rate among the fish and also to an unpredictable supply of fish for marketing. Furthermore, the diversion of food energy into gonad production is a financial waste and high food costs are among the limiting factors in salmonid farming. Total elimination of gonad in stock being reared for the table is therefore a desirable objective. Several methods have been suggested in the literature for the modification of gonad development in fish including surgical castration, hormone treatment and daylength control but, despite the attraction in developing a truly satisfactory method, all of these methods which have previously been considered tend to suffer from one or more disadvantages such as practicability on a large scale, expense etc. It is an OMPI ,< W1PO ,Λ< object of the present invention, therefore, to provide an improved method for the modification of gonad development in fish in order to produce fish which are not sexually mature. Accordingly the present invention comprises treating a fish to produce an auto-immune response therein which impairs the development and/or maintenance of a normal gonad system in the fish. It will be appreciated that the immune system of a fish differs considerably from that of a mammal, for example it contains only one major immunoglobulin rather than five. Therefore, although auto-immune responses are well known in mammals, it was previously by no means clear that it would even be possible to produce an auto-immune response in fish and, as far as we are aware, this is the first instance of any such auto-immune response being produced. The present invention is of particular application to teleosts (Teleostii) including Isospondyli and Ostariophysi, for example to salmoinids (or fish' of the family Salmonidae), especially to fish " of the sub family Salmonini containing the genera Salmo, Oncorhynchus, etc. Thus, it is of considerable interest in the treatment of salmon, for example both Atlantic and Pacific salmon, and trout, for example rainbow trout. The invention is however also of interest in relation to eels and especially to carp including Tilapia. Indeed, it will' be appreciated that the invention is applicable to a very wide variety of fish, both marine and fresh water, "BU RE OMPI although its benefits will be particularly marked with certain types as described above. The method may be applied to the fish at various stages in their growth, for example in the case of salmon and related salmonids as alevin, parr, smolt, grilse or even older fish. Clearly, however, it is preferable to treat the fish at as early a stage as is conveniently possible in their sexual maturation, although it has been found that the method of the invention may be used not only to prevent the development of gonad tissue but also lead to the retrogression " thereof. Although the invention is' of particular application to male fish in view of the greater problems posed therewith by early maturation, it is also applicable to female fish. The production of the desired auto-immune response in the fish is effected by inducing in the fish the presence of antibodies against gonad tissue. This may be done by administering such antibodies to the fish directly or, more preferably, by administering to the fish antigenic material which induces the formati-on of such antibodies. Such antigenic material may be derived from sperm or particularly testis gonad tissue in the case of males and ovarian tissue in the case of females, varying degrees of purification being possible including the use of simple extracts of gonad tissue and purified antigens. When treating a population of salmon or like fish of mixed sex, it may be convenient in view of the difficulties associated with the sexing of salmon either to treat each fish with a mixture of - B UREAU OMPl antibodies or of antigenic material deriving from both male and female fish or, if it is not desired to do this, to treat each fish with male derived antibodies or antigenic material in view of the lesser interest in the application of the method to female fish. It will be appreciated that the use of purified, or at least partially purified, antigens may be desirable to remove side effects arising from other antigens present as impurities in a crude preparation which may lead to an auto-immune response in other parts of the body. It is believed that the antigens of interest in the present context are surface antigens, particularly those of a water soluble nature, and these may be extracted by a variety of techniques including the use of both ionic and non-ionic detergents, hypo- and hyper-osmotic shock treatment and sonication of membrane fractions. The antigenic material may, if desired, be administered . together with an adjuvant, for example complete Freund's adjuvant (CFA), or rather more preferably in view of the intended use of the fish as food, with an adjuvant material ' which, in contrast to .complete Freund's adjuvant contains a killed or inactivated fish pathogen or pathogens. The latter alternative has the advantage that by using as the adjuvant a vaccine such as vibrio or furunculosis vaccine the additional effect is produced of providing protection against the infection in question. A further form of adjuvant material which may be considered is one of the algal extracts with potent adjuvant properties such as carrageenin, for example the kapa, lambda or particularly the iota form. Such extracts are usually water-soluble and are of particular value, therefore, for use in the hyper-osmotic treatment described hereinafter. It may also be desirable to enhance the effect of the antigenic material by using material from one species for administration to another, for example rainbow trout material in salmon, and vice versa and/or by the use of hooster doses of antigen (or antibodies). Further modifications include the solubilisation of antigens by various techniques, particularly for use in the hyperosmotic treatment described hereinafter, and the attachment of antigens to larger molecules to form immune complexes, for example to immunoglobulins such as rabbit immunoglobulin. Various routes may be used for administration of the antigenic material (or antibodies where applicable) including oral and parenteral, for example intraperitoneal and intra¬ muscular, routes. If desired, an initial administration by such a parenteral route of the antigenic material together with an adjuvant such as complete Freund's adjuvant may be employed, followed by a further administration of the antigen only by a similar route. In compositionsfor such a mode of administration the antibodies or antigenic material are made up in a physiologically acceptable carrier or diluent. This is usually of liquid form, being for example, isotonic saline or phosphate buffered saline. One alternative, very convenient, method of administration involves immersion of the fish in an aqueous hyperosmotic medium which contains, or is followed by immersion in an aqueous medium containing ,the antigenic material. It is believedthat this method involves take up of the antigens primarily through the lateral line system but also secondarily through the gills. It will be appreciated that this method is generally more convenient than other routes of administration but that it generally requires a somewhat purer and more fully water soluble antigenic material. Various compounds may be used to produce the hyperosmotic solution including inorganic salts, for example sodium bicarbonate and chlorides such as magnesium, calcium, potassium and especially sodium chloride, sugars such as sucrose and dextrose, urea etc. The amount of the compound is selected to give an appropriate osmolarity to the solution, for example in the range from about 400 to 1650 milliosmoles or possibly even higher, for example up to about 2500 or 3500 milliosmoles or even more, although values of about 1650 milliosmoles are preferred. Thus 10 per cent w/v urea and 5 * 23 per cent w/v sodium chloride each give solutions of 1650 milliosmoles. A •two stage procedure in which the fish is immersed first in the hyperosmotic solution and then in the solution containing the antigenic material has certain advantages such as avoidance of any interaction of the compound producing the appropriate hyperosmotic effect with the antigenic material. The application of the present method to inhibit, or desirably to substantially eliminate, the gonad system of fish can have certain other advantages in addition to those indicated above. Thus, it will be appreciated that the present invention produces the very great advantage that once the need to harvest fish imposed by their achievement of sexual maturity has been removed it is possible to regulate their growth to a suitable size for harvesting merely through control of feeding so that farmed fish may be harvested at a time when wild fish are not available. Moreover, the present need to feed expensive pigmented food to all of a group of salmon in sea water in case of early maturity can be avoided, such food being fed only immediately prior to the selected time of marketing. A further advantage may be illustrated in relation to the present considerable interest concerning the introduction of Pacific salmon into the United Kingdom for farming purposes. This has led to considerable controversy regarding the possible competition which could arise with the native Atlantic salmon if these fish were to escape. The ability to gterilise the Pacific salmon at an early age would avoid any long term problem arising in the event of such escape. The invention is illustrated by the following Examples. EXAMPLE 1: Treatment of Salmon (Small group) A group of salmon parr of age 1 year plus and of mean length 10cm was used, it being known that some of the males would in the normal course of events mature in a period of about 2 months. One of these maturing males was killed and "the testes extracted. A whole testis was ground up and pushed through a fine mesh sieve to separate the cells. The testis jύREA∑ OMPI W1 > was then washed three times in phosphate buffered saline- (the first washing involving passage through a sieve of a mesh which accepts single cells in order to separate connective tissue etc., which is retained by the sieve), centrifuging after each washing to remove serum and other water soluble and suspendable solids, then decanting and resuspending for the next wash. The resulting pellet was suspended in phosphate buffered saline (3 ml) and mixed with complete Fr-βund''s" adjuvant (4ml) using an ultrasonic probe. The resulting emulsion (0.2ml) was then injected intraperitoneally into salmon parr on 13th October 1976. A follow-up injection of sperm in phosphate . " buffered saline (0.2 ml) was given four weeks later on 10th November 1976. A second set of salmon parr were kept in similar tanks as controls and received no treatment. The control group of salmon developed gonads and smolted in the normal way. Of the five treated fish, three survived until May 1977 when all had become smolts. These fish were killed and examined, material being fixed in 10% formal saline, embedded in hard paraffin wax, cut at f and stained in H and E, Mallory's trichrome and Una Pappenheim. The results obtained were as follows. Fish 1 This fish was male and both superficial examination and subsequent histology suggested that the fish had been maturing at the time of injection. The testes showed signs of degeneration and appeared to have adhered to the body wall. 0MP Histόlogical examination showed that massive degeneration of the testes had in fact taken place, together with oedoema and proliferation of fibrous tissue, neighbouring muscle tissue not being damaged. Staining with Una Pappenheim showed many pyroninophyllic cells; these being plasma cells associated with antibody production in immune reactions. ■ No germ cells could be seen on the section as would be expected in normal male stnolts which had been mature in the preceding Autumn. (The state of degeneration seen in the testis was similar to that seen in the pancreas in conditions such as acute infectious pancreatic necrosis where the pancreas degenerates and does not recover:) . Fish 2 When this fish was dissected it appeared that the gonads were those of a male smolt which had not matured previously, i.e. they comprised two long thin strands lying in the dorsal part of the body cavity. However, histological examination showed that the testes had in fact responded to the injection. Compared with a normal smolt testis there was an increase in fibrous tissue and in tfrepresence of plasma antibody-producing cell, and a lack of germ cells. Histological examination of spleen, liver and kidney from this fish showed: no abnormalities. Fish 3 Gross and histological examination showed this fish to be a female fish which was developing in the manner of an untreated smolt. -BUREAU OMPI " It will be seen from the results obtained upon examination: of the three ish that degeneration of both immature and. matures male gonad was brought about by injection of a gonad/adjuvant preparation whilst no other organs were apparently effected, and smoltification and growth were also apparently unaffected. At the same time, development of the female gonad was not affected. EXAMPLE 2: Treatment of Salmon (Large group) A group of 650 salmon parr of an age 1 freshwater plus 1 sea water and of a size range 30 - 50 cm. length was used, the fish being both male and female. The fish were divided randomly into seven sub-groups which were treated as follows on l4th March 1978, the treatments being administered after benzocaine anaethesia by intreperitoneal injection with a 23 gauge hypodermic needle, and subsequently ' marked by freeze branding for subsequent identification of the differently treated groups (saline used was phosphate buffered): 1) no treatment (50 fish) 2) 0.2ml of 0.85% w/v saline (50 fish) 3) 0.2ml of an emulsion o 0.85% w/v ' saline -in CFA (l/2 v/v) 4) 0.2ml of 0.85% w/v saline containing 20mg/ml iota carrageenan (X52 Augygel; batch No. 3557) ( 0 fish) 5) • 0.2ml ofO ' .85%w/v salinecontaining 10% v/v gonad extract prepared from tissue as described in Example 1 but using 50% male:5P% female gonad tissue, the latter (ovaries) being extracted in'the same way as male testis * ' (150 fish) " B URE 0MPI 6) 0.2ml of an emulsion consisting of gonad extract prepared as for (5), 0.85% w/v saline and CFA (IO/25/65 v/v/v) (150 fish) 7) 0.2ml of 0.85% w/v saline containing 10% v/v gonad extract prepared as for (5) and 20mg/ml of iota carrageenan (X52 Augygel; Batch No. 3557) (10 ish). On 12th April 1978 all fish were identified according to their previous treatment by means of their marking and were weighed and measured. The first hundred fish of each of the groups 3 -, 6 and 7 were treated with 0.2ml of 0.85% w/v saline containing 10% v/v of gonad extract prepared as for (5) and were then marked to identify them. On 9th May 1978, all fish were identified, weighed and measured as before and the first fifty of the doubly treated -fish of groups 5, 6 and 7 were treated with 0.2ml of 0.85% w/v saline containing 10% v/v of gonad extract prepared as for (5), then marked to identify them. On 26th July 1978, a small number of fish in each of the sub-groups were measured and weighed, then killed and examined. The results obtained on 12th April and 9th May were used to calculate a mean length and mean weight for each sub-group and from these figures to calculate a condition factor (weight/ cube of length). In the case of the measurements made on 9th May a figure for daily growth in terms of both weight and length was calculated. The'data thus obtained are shown in Tables 1 and 2. It will be seen that the treatments did -BU EAU OMPI . WIPO Λ> not produce any statistically significant adverse effect on the growth of the fish during the two months following treatment. (Slight variation in the number of fish recovered from expected figures, especially in sub-groups 1, 2 and 6, arose from minor errors in counting of the original numbers in individual groups and/or in subsequent identification of fish according to individual groups.) On superficial visual examination of the dissected fish which were killed on 26th July it was found that atrophy of the gonads had occurred in all male fish of sections (b) and (c) of the sixth (CFA) sub-group. Similar atrophy was not identifiable in the absence of histological examination, which still remains to be carried out, in female fish of the sixth group or in fish of the fifth and seventh groups. Processing of the results' on the length and weight of the fish to provide data such as is presented for the 12th April and 9th May results similarly remains to be carried out. However, an initial examination suggests that there was again no significant difference in this- respect between the various groups. It will be appreciated that the advantages in relation to growth dependent upon failure to achieve sexual maturity would only be expected to emerge at a later stage of development of the whole group of salmon. Table 1; Data for 12th April 1978 Group Number Mean Mean Condition (Treatment) Recovered Length Weight Factor cm. gm• gm. cm Untreated 45 40.74 " 810.67 1.20 Saline 52 39-78 763-84 1.21 Saline + CFA 50 40.38 801.00 1.22 Saline + 50 40.33 791.60 1.21 carrageenan Saline + total 148 40.56 799.73 1.20 gonad extract (a) 1 - 100 39-99 750.4 1.17 total 148 (b) 101-148 41.75 902.5 1.24 Saline + total 165 3 " .-67 ' 748.85 1.20 gonad extract + CFA a) 1 - 100 39.90 758.20 1.19 b) 101-165 39.31 734.46 1.21 Saline + gonad s . , _,■ , ,„ _-_. extrac.t. + a total 146 40.69 815.07 1.21 carrageenan a) 1 - 100 40.43 78 . 6 1.19 b) 101-146 42.28 882.95 1.21 Table 2; Data for 9th May 1978 Group Number Mean Mean Condition Daily (Treatment) Recovered Length Weight Factor GGrroowwtthh cm. gm. gm.cm RRaattee % _ Day Length Weight Untreated 54 42.71 956.85 1.23 0.18 0.6 Saline 49 41.55 872.24 1.22 0.16 0.49 Saline + CFA 49 42.08 914.90 1.23 0.15 0.49 Saline + 47 42.06 915-53 1.23 0.16 0.54 carrageenan Saline + total 148 42.34 932.7 1.23 0.16 0.57 gonad extract a) 1 - 50 42.07 893.80 1.20 r b) I"- 98 41.88 883.26 1.20 0.17 0.6l c) 98-148 43.23 1029•6 1.27 Saline + total 159 41.44 872.57 1.23 0.16 0.57 gonad extract + CFA a) 1 - 50 - 41.87 904.40 1.23 b) 1 - 91 4l.6θ 889.56 1.24 0.15 0.59 c) 92 -159 41.22 849.85 1.21 Saline + total 146 42.38 933.84 1.23 0.15 0.51 gonad extract + carrageenan a) 1 - 50 42.08 915.00 1.23 b) 1 - 97 42.07 903.40 1.21 0.15 0.52 c) 98 - 146 43.OO 994.08 1.25 EXAMPLE 3; Treatment of Rainbow Trout A mixed sex group of rainbow trout of age 1 year plus was used. The fish were divided randomly into six sub-groups. One of these sub-groups received no treatment and was used as a control. The other sub-groups were treated on 1st March 1978 by intra-peritoneal (i.p.) injection with a gonad extract prepared from male rainbow trout in a substantially similar manner to that described for salmon in Example 1, no follow-up injection with sperm..being used however. The first of these sub-groups received a mixture of 0.05 tal of the testis suspension together with 0 * 1 ml of complete Freund's adjuvant and 0.08 ml of an extra cellular product (E.C.P.) isolated from cultures of Aeromonas salmonicida whilst the second received a mixture which was similar as regards two components but which instead of the E.C.P. contained 0.1 ml of isotonic 10 phosphate buffered saline containing 10 washed and formalin killed Aeromonas salmonicida cells. The remaining three sub-groups were treated on the same date with the same gonad extract by a hyperosmotic infiltration (h.i.) technique, the fish being immersed for 2 minutes in a bath containing 8% w/v of NaCl and then for 2 minutes in a second bath containing the gonad extract. The different treatments accorded to each sub-group involved the use of a second bath of 500 ml of water containing 4 ml of the testis extract in each case, together with 2 ml E.C.P. for the - 16 - 11 first group, 20 ml. E.C.P. for the second group, and 10 washed and killed Aeromonas salmonicida cells for the third group. Eleven weeks after treatment the fish were killed on 17th May 1978 and their serum tested for sperm agglutinating factor against sperm obtained by stripping a ripe male rainbow trout and washing the isperm three times in isotonic phosphate buffered saline. The test employs the usual slide agglutination technique with doubling dilutions to determine the greatest level of dilution at which agglutination is still present. Table 3 shows the results obtained for the various sub-groups of fish in the slide agglutination test (zero indicating complete lack of agglutination at the concentrated level) and it will be seen that the fish of all six treated sub-groups showed sperm agglutinating factor in their serum. Table 3» Sperm Agglutinating Factor in Rainbow Trout Serum Sub-Group Number in Group Agglutination Levels Unvaccinated 2 0 5 0 1 (i.p.) 1 1:4 2" (i.p.) 2 ' 1:4 ; 1:4 1 (h.i.) 2 1:4 ; 0 2 (h.i.) . . 2 1:8 ; 1:2 3 (h.i.) 2 1:4 ; 1:4 <_

Claims: CLAIMS 1. A method of treating fish which comprises inducing in fish the presence of antibodies against gonad tissue thereby producing an auto-immune response therein which impairs the development and/or maintenance of a normal gonad system in the fish. 2. A method according to Claim 1, wherein the fish is of the teleost group. 3. A method according to Claim 2, wherein the fish is a salmonid. 4. A method according to Claim 2, wherein the fish is a salmon or trout. 5. A method according to any of Claims 1 to 4, wherein the fish is a male. 6. A method according to any of the preceding claims, wherein the antibodies are induced in the fish by the administration of antigenic material derived from fish gonad tissue. 7. A method according to Claim 6, wherein the antigenic material is administered parenterally by injection. 8. A method according to Claim 6, wherein the antigenic material is administered through immersion in a medium containing the antigenic material, simultaneously or subsequently to treatment with a hyperosmotic medium. 9. A method according to' any of Claims 6, 7 and 8, wherein the effect of the antigenic material is enhanced by the use of an adjuvant. - BU AU OMPI „ IPO Λ 10. A method according to Claim 9 ι wherein the adjuvant comprises one or more killed or inactivated fish pathogens. 11. A method according to Claim 9, wherein the adjuvant is a Freund's adjuvant. 12. A method according to any of Claims 1 to 5» wherein the antibodies are induced in the fish by the direct administration thereof. 13. A method according to any of Claims 6 to 12, wherein the effect of the antigenic material or the antibodies is enhanced by the use of a booster treatment.- 14. A method according to Claim 1 substantially as described. in Example 1. 15- A method according to Claim: 1 substantially as described in Example 2. 16. A physiologically acceptable composition for the treatment of fish to impair the development and/or maintenance of a normal gonad system in the fish which comprises .as an active ingredient thereof antigenic material derived from fish gonad tissue. 17. A composition according to Claim 15 which comprises an adjuvant. 18. A composition according to Claim 17, wherein the adjuvant is a Freund's adjuvant. 19. A composition according to Claim 16, wherein the adjuvant comprises one or more killed or inactivated fish pathogens. 20. A physiologically acceptable composition for the treatment of fish to impair the development and/or maintenance of a normal gonad system in the fish which comprises as an active ingredient thereof antibodies against gonad tissue. 21. A composition according to any of Claims 16 to 20, wherein the fish is of the teleost group. 22. A composition according to Claim 21, wherein the fish is a.salmonid. 23« A composition according to Claim 21,, wherein the fish is a salmon or trout. 24. A composition according to any of Claims l6 to 23 in unit dosage form. 25. composition according to Claim 16 substantially as described in Example 1. 26. A composition according to Claim 16 substantially as described in Example 2. 27. A method according to Claim 1 which employs a composition according to any of Claims 16 to 24. 28. Fish whenever treated according to the method of any of Claims 1 to 15 and 27. -BUR A Γ OMPI

Inventors: ELLIS A, HOLLIDAY F, LAIRD L, WILSON A
Applicants: ELLIS A, HOLLIDAY F, LAIRD L, NAT RES DEV, WILSON A, NATIONAL RES DEV CORP
Application year: 1978
Main classification: A61K39
IPC codes: A01K61, A61K39