In-vivo Methods of Demonstrating the Efficacy of Fish Skin Protective Treatments
D. Signor; J. White; N.R. Smith
Department of Biological Science, California State University
Hayward, Hayward, CA
Most fish species have, in association with the epidermis, an outer
cuticle layer comprised of a mucopolysaccharide coat that appears to be secreted by both
Malphigian and goblet cells (Bullock and Roberts, 1974; Whitear, 1970; Whitear and Mittal,
1984). This mucoid coat is useful for escaping capture, and protection against bacterial and
parasitic infections. This protection is both mechanical: by providing a slimy barrier to
the epidermis; and biochemical: due to the presence of inhibitory substances in the mucous
that are anti-parasitic and anti-microbial. It has also been suggested that these mucoid
secretions, when dissolved in the water, decrease the friction of turbulent water, thereby
increasing locomotory efficiency by reducing drag (Bullock and Roberts, 1974). For all these
reasons, loss of this protective coat through handling, intensive crowding, and poor health
will be detrimental to the fitness of the fish, and the exposed epidermis susceptible to
attack by a variety of fish parasites and bacteria.
There are several commercially available products that have been
developed to serve as an artificial source of mucous protection. These products claim to
bind to the epidermis, replacing the natural mucous in areas where it has been removed. Such
water treatments are presently being used by both hobbyists and aqua culturists. However,
due to increased stringency in federal regulations imposed on the aquaculture industry, it
has now become necessary for the manufacturers of fish skin therapeutics to prove the
efficacy of their products. Two such protective treatments, Polyaqua and Novaqua were
subjected to a series of in-vivo experiments using the channel catfish, Ictalurus punctatus
in an effort to demonstrate product binding to the epidermal layer. These products are
designed to mimic the natural mucous layer of the fish skin, and as a result, are difficult
to distinguish from natural fish exudates.
Polyaqua and Novaqua were mixed with various markers, including
polystyrene latex beads, and iodine, and applied to the water according to manufacturer's
recommendation. Detection of the tagged products was attempted by examination of the catfish
epithelium using transmission electron microscopy (TEM), scanning electron microscopy (SEM),
and x-ray microanalysis. The channel catfish was chosen because it lacks scales and pigment
granules that may make examination and interpretation more difficult. Removal of the natural
mucous layer was imperative in order to determine if the products had bound to the
epithelium, and was attempted by several methods including physical wiping of the skin
(Shiomi, et al., 1988; Marshall, 1978; Hart and Oglesby, 1979), and application of 70%
ethanol. Polystyrene latex beads mixed with the product appeared to be the most reliable
marker for the detection of the product, but the difficulties encountered during
experimentation raised the question: How does one accurately approach proving the efficacy
of fish skin protective products? An in-vitro approach using Fat Head Minnow (FHM)
epithelial cell cultures and covalently linked fluorescent markers that can be visualized
in-situ using fluorescent microscopy may be the answer.
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