Organ And Tissue Indicators Composition of Blood

Changes in the composition of blood are used commonly to assess the clinical condition of human and non-human animals. Blood parameters have the advantage that the tissue is relatively easy to sample, and under some circumstances, samples can be taken without sacrificing the animal. However, blood characteristics differ markedly among species and even within a species there are significant seasonal changes. Consequently, prior knowledge of the normal range of values for a given species under given situations is needed. Not surprisingly, because of the large number of living fish species, there are still only relatively few data of blood composition, particularly of fish taken from the wild. Thus before these measures can be used to assess physiological disorders, it may be necessary to undertake preliminary studies of normal states, or to run parallel studies of impacted and non-impacted populations (stocks) of the same species.

It is beyond the scope of this chapter to deal with all aspects of blood composition that can be used as biomarkers of dysfunctional physiological conditions, but some of the more routine indicators are identified in the following section. Changes in white blood cells form an important part of the evaluation of infectious diseases in fish; this aspect of clinical evaluation has been dealt with in previous volumes in this series, and will not be considered at length here. However, it must be remembered that even for non-infectious conditions the competency of the immune system might be affected (see Chapter 11), and thus secondary infectious conditions might, in some instances, be present. The exposure of animals to some classes of toxicants, the imposition of inappropriate temperature regimes and the deprivation of key nutrients are all known to suppress the immune system of fish.

The percent red blood cell content (haematocrit) and haemoglobin content of whole blood, and red blood cell fragility can be applied readily to assess the overall condition of the animal, as can some aspects of blood coagulation. Also, changes in the composition of plasma (or serum) are used widely in assessment of the health of an animal, including fish, and as a measure of the response of the animal to environmental stressors.

Blood plasma proteins are central to several vital blood activities, including haemostasis and blood coagulation, vitamin and hormone transport, and specific immunity to pathogens. Plasma also contains significant amounts of active and inactive enzymes. Low total plasma protein is a first line indicator of a general problem, but it cannot be effectively interpreted without further analysis. Total plasma triglyceride and cholesterol levels may provide an indirect measure of the availability of these nutrient reserves; however, post-prandial changes in the delivery of these nutrients to the blood may confound interpretation. This may be particularly problematic in field studies, when the time of feeding is not known.

To overcome some of the problems associated with the interpretation of total plasma nutrient concentrations, measurements of their metabolites are usually the preferred means of assessing the nutrient status of an animal. Concentrations of metabolites in plasma (and tissues) are functions of the rate of synthesis, the rate of release from tissues into the blood, and the rate of degradation and clearance from the blood; therefore, although the absolute concentration of a metabolite may be constant the turnover may vary considerably. Since the determination of the turnover of metabolites usually involves the use of labelled metabolites, the method is not readily applied to field studies, thus the use of plasma concentrations of metabolites is often the measurement of choice. Since communication between tissues, delivery of substrates as energy sources and removal of waste products all occur via the blood, blood metabolite levels are a potentially useful index of the health of fish. Some of the most commonly measured metabolites are considered below.

Plasma glucose concentration is frequently measured although its value for the assessment of metabolic dysfunction is questionable. Plasma glucose levels vary with species, size, age, sex, nutritional and reproductive status (see McDonald and Milligan, 1992, for review). In addition, stress-related phenomena, including hypoxia, handling, exercise and disease, bring about changes in plasma glucose levels. If changes in plasma glucose concentration are to be used as an indicator of environment-induced metabolic dysfunction, these factors have to be carefully controlled; since this is frequently impossible in field situations, these measures may be of limited diagnostic value. Similarly, although plasma lactate levels are indicative of oxygen limitation (environmental hypoxia, tissue ischaemia, etc.), they are dependent on the sampling procedures used, and the physical exertion by the fish at the time of sampling is likely to result in elevated plasma lactate levels (McDonald and Milligan, 1992).

Concentrations of free amino acids in the plasma have also been used for health assessment of fishes. Fish plasma contains some free amino acids (e.g. taurine) and related compounds (methylamines) not found in plasma protein. It is assumed (Wilson, 1989) that fish have the same ten essential amino acids (arginine, histidine, leucine, isoleucine, lysine, methionine, phenylalanine, threonine, tryptophan and valine) as in other vertebrates. The levels of essential amino acids could, in principle, be used to assess the nutritional status of wild populations of fish provided enough background information related to the normal levels of these amino acids is available for a given species. The large number of free amino acids in the plasma makes analysis of their levels a very powerful tool for the diagnosis of dysfunctional conditions. For example, the presence of 3-methylhistidine in the blood is indicative of protein degradation since histidine is only methylated in proteins and is released by proteolysis. The free amino acids in the plasma of fishes are influenced by a variety of factors that are only now being investigated. Measurement of certain plasma-free amino acids can be accomplished by using enzymatic methods (Bergmeyer, 1963) but the versatility and ability to simultaneously detect dozens of amino acids makes automated amino acid analysis using HPLC methods a preferred option.

As with the amino acids, changes in the concentration and proportion of non-esterified fatty acids (NEFAs) in the plasma of fishes have been used as indices of the health of wild populations of fish. The types of fatty acids in the non-esterified fraction of blood are associated with different metabolic processes. They may serve as metabolic substrate components of membranes and as precursors for prostaglandins. Consequently, the identification of the full NEFA complement yields considerable information about the animal's metabolism. Since absolute levels of NEFAs may vary considerably, an important strategy is to factor out such confounding factors by using ratios of fatty acids involved in one aspect of metabolism to those involved in other pathways. Such types of analyses have been used to assess the health of lake sturgeon (Acipenser fulvescens) (McKinley et al., 1993), and the metabolic stresses associated with the terminal spawning migrations of sea lamprey (Petromyzon marinus) (LeBlanc et al., 1995) and sockeye salmon (Ballantyne et al, 1996).

Plasma enzyme levels have also been used to identify tissues influenced by a stressor (for review see Hille, 1982). The presence of these enzymes is sometimes indicative of tissue damage. McDonald and Milligan (1992) have summarized the tissues of origin and the factors influencing the levels of these plasma enzymes. Application of these methods to wild or cultured populations of fish is possible but season (Melotti et al., 1989; Lenhardt, 1992) and temperature (Grigo, 1975a,b) influence plasma enzyme levels. Moreover, enzyme activity of plasma (and tissues) is rapidly lost unless the sample is stabilized (usually by freezing) immediately on collection.

An additional difficulty is that most of the published work relating to measurement of enzyme activities pertains to mammals. For many enzymes in many fish species it is necessary to determine the optimal substrate saturation conditions for the enzyme in question. (See below, the discussion of enzyme measurements in organs and tissues.)

Changes in plasma hormone concentrations are widely employed as indicators of the physiological condition of fish. Changes in the levels of hormones associated with reproduction are useful indicators of impaired reproductive function (see above). 'Stress' hormones, such as cortisol, are commonly used as indicators of acute or chronic responses to environmental stressors (see Chapter 11). In addition, hormones that are involved in the regulation of metabolic functions may also lend themselves to diagnostic assessment. For example, in some fishes, a reduction in food consumption (either enforced or by choice) is followed, within a few hours, by a reduction in the production of the thyroid hormone triiodo-L-thyronine (T3), and thus in a fall in blood T3 content (Farbridge et al., 1992; Leatherland and Farbridge, 1992). We have, therefore, employed commercially available assays to measure plasma T3 concentration to assess the nutritional condition of fish species held in culture (e.g. Bandeen and Leatherland 1997a,b). Plasma growth hormone (GH), insulin-like growth factor (IGF), insulin, somatostatin and glucagon concentrations have similar value in the assessment of physiological condition (Wendelaar-Bonga, 1993). However, the availability of these assays for application to fish is limited to the small number of laboratories that have developed the necessary assay systems.

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