Bill Woodward

Dietary vitamin requirements of cultured young fish, with emphasis on quantitative estimates for salmonids

Abstract The majority of currently accepted estimates of vitamin requirements for salmon are astonishingly high. These estimates are, for the most part, based on the use of the classic H440 purified diet. This diet provided the seminal results pertaining to water-soluble vitamin deficiency signs in salmonids, especially Pacific salmon. The H440 diet, however, is highly water-soluble and thus inappropriate for precise estimation of water-soluble vitamin requirements. Unfortunately, this is now generally overlooked. Improved purified diets (such as that developed in Guelph, Ontario, during the 1970s) are high-fat, steampelleted formulations which minimize leaching losses and which support good growth rates in rainbow trout ( Oncorhynchus mykiss ). This species is particularly suitable for studies of nutrient requirements because of its voracious feeding behaviour in captivity. Weight gain is arguably the best single index of vitamin requirement on which to base diet formulation practice. On the basis of this criterion, the dietary requirements for most water-soluble vitamins do not differ substantially when comparing the trout and diverse warm-blooded species. Interspecies similarity also is apparent regardless of the way in which vitamin requirements are expressed (i.e. on a dietary weight or energy basis), regardless of strain-specific growth potential, and (possibly except for vitamin E) regardless of water temperature. From the standpoint of phylogeny, therefore, results obtained using rainbow trout should be considered generally applicable to other salmonid species. Exceptions to this generalization may include vitamin B 12 as well as the vitamin-like nutrients, choline and myo-inositol. Vitamin C is a special case for which a precise estimate of minimal requirement has awaited discovery of a stable, biologically available form. Attention is now focused on ascorbate phosphate in this regard. More precise information is needed in relation to the minimal dietary requirements of salmonids for the fat-soluble vitamins, as well as for vitamin C and the vitamin-like compounds choline and myo-inositol. Ultimately, it may prove useful to define a maximum dietary requirement for nutrients such as vitamin K, vitamin B 12 , choline and myo-inositol for which significant and variable non-dietary sources may exist. Minimal dietary requirements are defined by means of diets containing large surfeits of all components except for the nutrient which is under investigation. It will be important to determine whether a diet containing all the vitamins at their minimal requirement level (defined as indicated) is, in fact, complete for salmonids or other species.

Dietary arginine requirement of young rainbow trout (Oncorhynchus mykiss)

1. Two growth trials were conducted with young rainbow trout (Oncorhynchus mykiss) to determine the dietary arginine requirement under conditions of rapid weight gain at 15 degrees C. 2. The growth requirement does not exceed 4.2 g arginine/16 g dietary nitrogen and, thus, is much lower than the value of 6.0 g arginine/16 g dietary nitrogen presently listed by the NRC for Chinook salmon and widely applied to all Salmonids. 3. Comparison of the present results with the arginine requirement of the chick reveals remarkable similarity despite the phylogenetic distance between the two species, and demonstrates the need to re-evaluate, as anomalously high, the presently-accepted value for Chinook salmon.

Epithelial and pillar cell replacement in gills of juvenile trout, Salmo gairdneri Richardson

Young rainbow trout, Salmo gairdneri (Richardson) were injected intraperitoneally with tritiated thymidine, and killed at intervals between 2 hr and 16 days after inoculation. Labelled epithelial cells were first detected autoradiographically along the base of gill lamellae. Epithelial cells proliferated here and then migrated toward the tips of the lamellae. Uniform labelling along the length of the filaments at the base of lamellae indicated that cells were dividing at a constant rate. Transverse sections of filaments showed that epithelial proliferation was also uniform across the base of the lamellae. The interior of the lamellae often had labelled pillar cells, indicating that these cells also divide. The high intensity of the label in animals killed 16 days after inoculation with tritiated thymidine suggests that division probably occurs slowly, less than once every 16 days.

Vomitoxin in diets for rainbow trout (Salmo gairdneri)

Abstract The present study was conducted to obtain information as to the response and sensitivity of rainbow trout ( Salmo gairdneri ) to diets containing vomitoxin-contaminated corn. Feed refusal occurred when diets contained 20 μ g/g vomitoxin or more, but the trout recovered rapidly when subsequently fed a diet containing no detectable toxin. Diets containing graded levels of vomitoxin, incresing from 1.0 to 13.0 μ g/g, caused progressively greater depression in 4-week liveweight gains of juvenile trout. The depression in weight gain ranged from 12% to 92% of the control value and resulted from an adverse effect on both feed intake and feed conversion efficiency. Emesis was not observed in this work. The results demonstrate that rainbow trout are highly sensitive to dietary vomitoxin.

Blood Corticosterone Concentration Reaches Critical Illness Levels Early During Acute Malnutrition in the Weanling Mouse

Acute (i.e., wasting) pediatric malnutrition consistently elevates blood glucocorticoid levels, but neither the magnitude of the rise in concentration nor its kinetics is clear. Male and female C57BL/6J mice, initially 19 days old, and CBA/J mice, initially 23 days old, consumed a complete purified diet either ad libitum (age-matched control) or in restricted daily quantities (mimicking marasmus), or they consumed a purified isocaloric low-protein diet ad libitum (mimicking incipient kwashiorkor). Serum levels of corticosterone were assessed by double antibody radioimmunoassay after 3, 6, and 14 days (C57BLV6J strain) or after 6 and 14 days in the genetically distant CBA/J strain. Age-matched control groups of both strains exhibited mean corticosterone levels of 5–30 ng/ml, whereas the acutely malnourished groups exhibited mean levels of this hormone that were elevated by more than an order of magnitude as early as 3 days after initiation of weight loss. This outcome was confirmed in a second experiment in which the serum corticosterone level of C57BL/6J weanlings was examined by competitive binding enzyme immunoassay 3 and 14 days after initiation of the dietary protocols. Therefore, deficits of protein and/or energy in weanling murine systems relevant to acute pediatric malnutrition elicit early elevations in blood glucocorticoid levels to a magnitude reminiscent of critical illness and multiple trauma. The key to this novel finding was an exsanguination method that permitted accurate assessment of the blood corticosterone level of the healthy, quiescent mouse. Overall, the results of this investigation provide a new perspective on the glucocorticoids as part of the early hormonal response to acute weanling malnutrition coincident with the shift toward catabolic metabolism and the initiation of depression in cellular immune competence.

INFLUENCE OF SEVERE PROTEIN DEFICIENCY AND OF SEVERE FOOD INTAKE RESTRICTION ON SERUM LEVELS OF THYROID HORMONES IN THE WEANLING MOUSE

Abstract Serum levels of thyroid hormones viz. total and free thyroxine (T 4 ) and triiodothyronine (T 3 ) were determined in protocols representing the two most common rodent models of severe protein-energy malnutrition (PEM). In one experiment male and female CBA/J mice were fed from 23 to 37 days of age either a nutritionally adequate diet in restricted quantities or an isocaloric low-protein formulation ad libitum. An ad libitum-fed control group was also included. In a second experiment a control and a low-protein group were fed from 23 to 63 days of age. Severe malnutrition was indicated by weight loss, by mortalities and, in the longer-term study, by the development of fluid accumulation. Mice fed the low-protein diet for 14 days exhibited serum total and free T 3 levels which did not differ from control values, while total T 4 levels were high. By contrast, the food intake restriction protocol (14 days) and the low-protein diet fed for 40 days each mimicked the human response to severe PEM insofar as serum total and free T 3 levels were reduced, in the present experiments, to values between 12% and 54% of control levels. The results show that both the food intake restriction type and (contrary to existing evidence) the low-protein type of rodent protocol for experimental PEM can be used to model the human thyroid hormone response to severe malnutrition.

Thymic Epithelial Cells of Severely Undernourished Mice: Accumulation of Cholesteryl Esters and Absence of Cytoplasmic Vacuoles

Abstract The thymic epithelium was compared in weanling male and female CBA/J mice when fed ad libitumand when subjected to severe food intake restriction for 14 days. The restriction protocol elicited predominantly a metabolic response to caloric deficit rather than to protein deficiency. Electron microscopy revealed intracytoplasmic accumulations of large, circular, homogeneously electron-dense profiles (with no limiting membrane) in a high proportion of cortical and medullary epithelial cells of thymuses from restricted mice, but not from controls. The electron-dense material was not preserved in the absence of osmium. Thin-layer chromatography (TLC) indicated elevated levels of free and esterified cholesterol, particularly the latter, in whole thymus extracts of restricted mice. Measurements of total cholesterol levels in the thymic extracts were consistent with the results obtained by TLC. In addition, cryostat sections of thymuses from restricted mice, but not from controls, exhibited numerous stained foci throughout the cortex and medulla when treated with oil red O (a general neutral lipid stain) or by the Schultz procedure which is specific for cholesterol. Collectively the results suggest accumulations of cholesteryl esters, together with some free cholesterol, as non-membrane-bound droplets in the cytoplasm of thymic epithelial cells of undernourished mice. It is also of interest that the lipid-laden epithelial cells exhibited none of the cytoplasmic vacuoles observed in controls and believed to be important in thymic hormone secretion. This work provides the first direct evidence of thymus epithelial abnormalities in severe protein-energy malnutrition.

Involution of thymic epithelium and low serum thymulin bioactivity in weanling mice subjected to severe food intake restriction or severe protein deficiency

The volume of epithelium in the cortex and in the medulla of the thymus was compared in four groups of weanling male and female CBA/J mice. Well-nourished controls (C), food intake restricted (R), and animals given a low-protein diet ad libitum (LP) were fed from 23 to 37 days of age. Baseline controls (B) were studied at 23 days of age. Epithelial volume fraction was estimated for each group by point-counting morphometry on electron micrographs. Other mice were used to obtain group mean estimates of thymic index (mg/g live weight) and volume fraction of cortex and medulla (light microscope-level point-counting morphometry). Cortical and medullary epithelial volumes were calculated for each animal examined by electron microscopy by obtaining the live weight and applying, in sequence, the group mean thymic index, an assumed thymic density of 1.0 mm3/mg, the group mean cortical or medullary volume fraction, and the measured cortical or medullary volume fraction for that animal. Serum thymulin bioactivity was also measured in C, R, and LP mice. The results reveal thymic epithelial involution in the two most common rodent models of malnutrition, and suggest that this may contribute to the low serum thymulin levels found in malnourished experimental animals and humans.

Relationship between serum zinc level and immunocompetence in protein-deficient and well-nourished weanling mice

Abstract Two experiments were performed in which three-week-old male and female CBA/J mice were fed ad libitum for two weeks either a control diet (18.6% protein) containing adequate levels of all nutrients or a lowprotein diet (Experiment 1: 1.6%; Experiment 2: 0.6%) adequate in all other nutrients. Two thymus-dependent primary immune responses to sheep red blood cells (SRBC), viz. the antibody (hemagglutinin) response and the low-dose delayed hypersensitivity response, were tested in each experiment. Mice fed 1.6% protein maintained weaning weight, but exhibited low thymic and splenic indices (weight relative to body weight), low serum protein levels and low serum zinc levels. These animals, however, displayed normal immune responses to SRBC. Mice fed 0.6% protein lost about 15–20% of their initial (weaning) weight during the 14-day experiment, and exhibited low thymic and splenic indices, low serum protein levels and low serum zinc levels. Both the antibody response and the delayed hypersensitivity reaction were depressed in these animals. Subcutaneous zinc injections raised the serum zinc level to nearnormal in mice fed 0.6% protein, but did not influence either immune response examined. By contrast parenterally administered zinc reduced the antibody response of well nourished mice to about 15% of normal, although the delayed hypersensitivity response was not affected in this group of animals. A negative correlation (R=−0.72, P

Elevated Bioactivity of the Tolerogenic Cytokines, Interleukin-10 and Transforming Growth Factor-β, in the Blood of Acutely Malnourished Weanling Mice

The main objective of this investigation was to determine the influence of acute deficits of protein and energy on the blood levels of interleukin-10 (IL-10) and transforming growth factor-β (TGF-β), physiologically the main anti-inflammatory and tolerogenic cytokines. In four 14-day experiments, male and female C57BL/6J mice, initially 19 days old, consumed a complete purified diet either ad libitum or in restricted daily quantities, or had free access to an isocaloric purified low-protein diet. A zero-time control group (19 days old) was included. In the first two experiments, serum IL-10 levels were assessed by sandwich enzyme-linked immunosorbent assay (ELISA) and bioassay. The mean serum IL-10 bioactivities were higher (P ≤ 0.05) in both malnourished groups (low-protein and restricted intake: 15.8 and 12.2 ng/ml, respectively) than in the zero-time and age-matched control groups (6.3 and 7.3 ng/ml, respectively), whereas serum IL-10 immunoactivity was high only in the restricted intake group (e.g., second experiment: 17.0 pg/ml vs. 5.4, 3.7, and 3.1 pg/ml in the zero-time control, age-matched control and low-protein group, respectively). The third and fourth experiments centered on plasma TGF-β immunoactivity (sandwich ELISA) and bioactivity, respectively. The ELISA revealed a high mean plasma TGF-β1 level (P < 0.05) in the low-protein group only, but TGF-β bioactivity (β1 isoform, although 15% β2 in the restricted intake group) was high in both malnourished groups (8.7 and 9.3 ng/ml in the low-protein and restricted groups, respectively) relative to the age-matched control group (0.5 ng/ml). Thus, metabolically distinct weanling systems mimicking marasmus and incipient kwashiorkor both exhibit a blood cytokine profile that points to a tolerogenic microenvironment within immune response compartments. A model emerges in which malnutrition-associated immune competence, at least in advanced weight loss, centers on cytokine-mediated peripheral tolerance that reduces the risk of catabolically induced autoimmune disease, but this is at the cost of attenuated responsiveness to infectious agents.