Introduction

The major role of the vertebrate gut is one of digestion. As Such, it is generally assumed that proteins are completely hydrolysed before absorption. However, histochemical studies have demonstrated that ingested protein may be absorbed by enterocytes of the fish mid-, and hind-gut in an intact form (Table 1). Furthermore, it is now clear that certain orally administered proteins and peptides enter the fish bloodstream in a bioactive state (Table 2). The precise route(s) by which such molecules are absorbed, however, remain unclear. But, the potential for exploiting this natural phenomenon is manifest. An ability to orally deliver these therapeutants to fish would minimise not only the costs and time associated with treatment, but also eliminate the need to anaesthetize and handle the animal. The availability of oral drug formulations would thus permit therapy of difficult to-handle, and easily stressed fishes.

A variety of recent papers have examined the mechanisms of macromolecule absorption (McLean & Ash 1987a; Ash & McLean 1989), or have reviewed the potential physiological significance of this phenomenon to fish (Vernier & Sire 1989; McLean & Donaldson 1990). The following will center upon the potential applications of macromolecule absorption to the aquaculture and ornamental fish industries and provide brief consideration of the problems which may be encountered in developing oral drug formulations for teleosts.

Production-Orientated Applications of Macromolecular Uptake

Recombinant DNA technologies have provided a means of producing specific proteins en masse, while the production of smaller peptides has been augmented by increasingly refined techniques of peptide synthesis. Such advances in biotechnology have resulted in therapeutic peptides and proteins becoming more widely available and also more prominent as a means of controlling production-orientated metabolic processes in cultured teleosts. Currently, three important production-related procedures may be isolated as potential candidates for oral drug therapy. Each process is equally applicable to ornamental as well as food fish culture operations; although benefits derived from such manipulations may differ for each industry.

Controlled Reproduction

Parenteral administration of maturational hormones has been successfully employed as a means of manipulating the latter stages of the reproductive cycle in a variety of teleosts. In commercial aquaculture operations, artificial induction of ovulation is necessary, since in most species, ovulation does not occur spontaneously. Such inductions have the added benefit of synchronizing maturation of broodstock. However, it has been demonstrated that stress caused by physical manipulation, and a variety of other factors, inhibit the ovarian response and cause follicular atresia in fish. Furthermore, many species of teleost are difficult to manipulate due to their size (eg. Pacific halibut Hippoglossus stenolepis [> 2 6 0 cm]; 1eaf - fish Monocirrhus polyacanthus [< 8 cm]) - The availability of an effective method of inducing ovulation, without the necessity of handling would, therefore, offer considerable advantage.

Table 1. Example species of teleost in which intestinal permeability to macromolecules has been demonstrated using the protein tracer horseradish peroxidase (HRP, Sol. vt. 40000 daltons)

An early indication as to the feasibility of delivering maturational peptides and proteins to fish, using the oral route, was provided by Tuchmann (1936), and Regnier (1938). These authors reported that orally administered pituitary preparations influenced the sexual development of the guppy (Girardinus guppii) and swordtail (Xiphophorous helleri) respectively. More recently, Suzuki et al. (1988) demonstrated that oral intubation of a crude extract of salmon (Oncorhynchus keta) pituitary induced ovulation in the goldfish (Carassius auratus). In addition, Suzuki et al. (1988) revealed that salmon gonadotropia (GtH) levels in the plasma of intubated goldfish increased markedly; whereas endogenous GtH levels remained fairly constant. From this information, the authors concluded that the induced ovulation and spermiation of goldfish occurred as a result of the passage of physiologically active salmon GtH into the goldfish bloodstream. However, the application of such procedures to inducing ovulation in commercially important teleosts is limited by the lack of availability of standardized fish pituitary preparations, since they are both difficult to collect and often exhibit variable potency.

Table 2. Peptides and proteins which have been demonstrated to enter the fish bloodstream in a biologically active state following oral administration.

An alternative to pituitary-derived hormones however, are the gonadotropin releasing hormone (GnRH) analogues. Some of these molecules are known to resist hydrolysis and thus appear to offer considerable potential as candidates for controlling reproduction in fish using the oral route. In fact, recent trials have demonstrated this to be the case. Thomas & Boyd (1989) fed spotted seatrout (Cynoscion nebulosus) shrimp which had been spiked with 0.2-2.5 mg/kg body weight Des Gly¹⁰ (D-Ala⁶)LHRH ethylamide. This procedure resulted in induced ovulation in this warm-water species within 38 hr of administration. Furthermore, these authors reported a mean fertilization and hatching success rate of 93.3% and 74 .6% respectively - More recently, Solar et al ( 1990 ) reported that oral intubation of the same LHRH analog to the sablefish (Anopoploma fimbria) also induced ovulation in this cold water, marine species. A priming dose of I mg LHRH/kg body weight, followed eleven days later by a secondary intubation of 0.5 mg LHRH/kg body weight, resulted in the induction of ovulation within 18 days of the first treatment. Mean percent fertilization success was variable (22.6+10.5%), but similar to that achieved when this analog was delivered by injection. These studies thereby provide important, although preliminary data with respect to the potential for the oral administration of maturational peptides as a means of influencing sexual development in fish. Future studies will, no doubt, provide further evaluation of the above procedures from a practical standpoint; while the endocrine response of coho salmon intubated with LHRH and LHRHA has already been characterized (Donaldson et al. 1989). Oral delivery of GNRH analog may thus become important when attempting to control the life cycles of commercially cultured, exotic rare, and endangered species of teleost, that fail to ovulate in captivity or are sensitive to physical manipulation.

Growth Acceleration

The benefits of accelerating the growth of teleosts are many. In commercial aquaculture and enhancement programmes, growth acceleration provides a means of compressing the time-span required for fish to obtain appropriate body mass prior to release, seawater transfer or harvesting. Also, larger sized ornamental fish are of greater value. Notable success has been achieved in accelerating the growth of teleosts with both topical and parenteral administrations of native and recombinant somatotropins (eg Down et al. 1989a; Schulte et al. 1989). But such methods of Administration (injection, immersion) may be stressful to the recipient, time-consuming to perform and technically and logistically demanding. An effective method of orally delivering somatotropins would overcome many of the above constraints. Preliminary experiments have provided a clear indication that bovine growth hormone, when administered either in the diet (Tuchmann 1936; Regnier 1938; Degani & Gallagher 1985) orally (McLean et al. 1990a) or rectally (Le Bail et al. 1989), gains access to the bloodstream in a physiologically ctive state, and significantly increases growth performance. Further work upon this phenomenon is however, required. Future experiments must be undertaken to determine, for each species, the optimum dose of somatotropin for a particular developmental stage. Further, it is now apparent that certain recombinant, analog forms of somatotropin are more potent in stimulating growth of coho salmon (Oncorhynchus kisutch) than natural forms (Down et al. 1989b). Some reports indicate species-specific responses to parenterally delivered growth hormone. Thus, Wilson et al. 1988 observed that while injection of channel catfish Ictalurus punctatus, with rbGH provided a significant dose-dependent increase in growth when compared to injected controls, the performance of an unhandled group was essentially similar to fish treated with lower doses of the rbGH. Furthermore, Wilson et al. (1988) demonstrated that the increase in weight of rbGH treated channel catfish was a result of increased fat deposition, suggesting that in this species rbGH therapy resulted in a stimulation of appetite, but not skeletal growth. Other problems specific to the oral administration of somatotropins are considered further in Section III.

Vaccination

Relative to the alternate methods of vaccination (immersion, injection), oral immunization would provide not only a nonstressful method of vaccine presentation to fish, but also a saving in labour. The first successful oral immunization of fish was accomplished 50 years ago (Duff 1942). Since that time many studies have re-examined the potential for oral vaccination. However, while some have reported remarkable success, others have yielded less promising results. Yet, while it is evident that parenteral means of immunization are more effective than oral vaccination procedures, the convenience that the latter method of bacterin presentation offers, makes the study of this particular aspect of macromolecule absorption highly valuable.

The apparent failure of oral bacterin delivery in many trials has generally been ascribed to the destruction of the antigenic component by the digestive secretions of the gut. Support for such a contention is derived from the studies of Johnson & Amend (1983). These workers demonstrated that immunization of sockeye salmon Oncorhynchus nerka with a single rectal intubation of a Vibrio anguillarum bacterin, followed 59 days later with a water-borne challenge, resulted in 100% protection, while 55% of the control animals died. The results of Johnson & Amend (1983) led them to conclude that "for oral vaccination to be successful, a method of protecting the antigen through the upper part of the intestine must be developed". Such a deduction, which is consistent with the conclusions of others, reflects one of the major problems relating to the oral delivery of not only vaccines, but also other bioactive molecules. However, Lillehaug (1989) reported that oral immunization of rainbow trout Oncorhynchus mykiss with a Vibrio bacterin resulted in 97.8% survival following "seminatural" challenge; whereas only 52.8% of control unvaccinated animals survived. Such results suggest protection of certain bacterins from the gastrointestinal (GI) secretions may not therefore be imperative. But, it is noteworthy that to date, conspicuous success in oral immunization of fish has only been secured using Vibrio bacterins.

Notwithstanding the latter observations, the applicability of oral systems of drug delivery will rely heavily upon a treated individual receiving sufficient quantities of a specific drug(s). Practical problems may be encountered therefore, in that fish may not ingest the same amount of a medicated feed. Although this may be less of a difficulty with the treatment of ornamental fishes, this, and other problems (e.g. drug leaching, drug stability etc) must be taken into account when calculating percent drug incorporation into feed (if indeed, the feed is to be the desired vehicle for the delivery for a specific therapeutant).

Problems in the Delivery of Bioactive Peptides and Proteins

There can be little doubt that the fish gut is naturally permeable to orally ingested macromolecules (Tables 1 & 2). However, the application of this natural phenomenon to both ornamental and commercial fish rearing has jet to be implemented. This is because far greater quantities of therapeutant must be delivered using the oral route to achieve the desired effect, than is necessary following other methods of administration. A prerequisite to the full exploitation of this phenomenon therefore, is the design of an effective and reproducible method drug delivery. Certainly, a greater understanding of the processes of teleost digestion and absorption would speed the development of such drug delivery or carrier systems, but it is also possible that successful oral therapy can be realised with the application of knowledge that is currently at hand.

A wide variety of physica1, chemical and immunological components of the fish gut, acting either alone, or in concert, severely restrict the absorption of orally administered macromolecules (reviewed in McLean & Donaldson 1990). It is these same components that place limitations upon the design of an effective oral drug delivery system, since each, or several of these elements, must be countered when attempting to protect or enhance the delivery of a therapeutant. In order to enhance the GI absorption of a specific drug, a variety of schemes may be contemplated, the most obvious of which is to maximize the delivery of a drug to that segment of the gut which is thought to be most active in its sequestration. Thus, as suggested by Johnson & Amend (1983), microencapsulation, enteric coating, or polymer entrapment may provide the means by which particular peptides, proteins or vaccine components may be protected during passage through the upper regions of the GI tract. Such technologies have recently been applied to the oral delivery of Vibrio vaccines for rainbow trout (Lillehaug 1989). But, neither the entrapment of the Vibrio bacterin in an acid resistant film coating, or its incorporation into a matrix consisting of heavily degraded fatty acids, provided augmented protection when compared to animals in which the bacterin was delivered without such 'protection'. Nevertheless, such methodologies may prove useful in the delivery of acid sensitive peptides and proteins.

Alternatively, or in addition to the above procedures, it may be possible to include in such formulations inhibitors of the major extracellular digestive enzymes. Preliminary studies in which such techniques were employed (McLean & Ash 1990) demonstrated that the coincident oral delivery of horseradish peroxidase (HRP) and soybean trypsin inhibitor (SBTI) to rainbow trout resulted in an increased presence of HRP within the liver and spleen, relative to non-SBTI treated controls. Another approach to protecting orally delivered therapeutants may be to coadminister the drug with antacids. Since elevation of gastric pH results in inhibition of pepsinogen secretion, then antacids might also be utilised to protect acid-sensitive drugs from the action of pepsin. Application of such stratagems have been successfully employed in the oral delivery of Vibrio bacterins to rainbow trout (Anders 1978), rbGH to coho salmon (McLean et al. 1990) and LHRHa to sablefish (Solar et al. 1990).

Assuming that a therapeutant can be delivered to the site(s) of absorption in the GI tract, at maximal concentrations, it still remains for the molecule to gain access to, and avoid proteolysis in, the enterocyte, before entering the circulation. One approach which could potentially enhance the uptake of a drug would be its codelivery with a surface-active agent (SAA). SAAs might be used to temporarily decrease the physico-immuno-chemical barrier to absorption imposed by epithelial mucus. However, some authors (Suzuki et al. 1988) have reported that a SAA which had been successfully employed in enhancing the absorption of drugs in mammals, when coadministered with GtH remained ineffective in goldfish. However, other SAAs have been successfully utilized in the delivery of LHRHA (Solar et al. 1990) and rbGH (McLean et al. 1990).

Although by no means complete, the above studies all provide an indication of the potential success that may be gained in employing such methodologies. Such techniques deserve future consideration when evaluating means of controlled, quantitative and reproducible delivery of production-orientated therapeutants to both commercially important and ornamental species of fishes. Due consideration must also be given to the design of an effective drug carrier system. The development of such a system must take into account many parameters including: size, shape, palatability, stability (both in storage and within the aquatic environment), general ease of use, economy of production and incorporability into the feed (if desired). Ideally, the carrier should be equally applicable to a variety of drugs. Of particular importance is the ease with which size may be manipulated for use throughout the entire production cycle of a species. Information upon the dispersal of the drug(s) within the carrier matrix and their dissolution within the gut and subsequent uptake and action(s) must also be considered. Finally, any such formulations must meet or preferably exceed the applicable regulations for safety, efficacy and lack of environmental impact.

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