# Daily variations in plasma cortisol levels of individual female rainbow trout Salmo gairdneri: Evidence for a post-feeding peak in well-adapted fish

C. Bry

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C. Bry. Daily variations in plasma cortisol levels of individual female rainbow trout Salmo gairdneri: Evidence for a post-feeding peak in well-adapted fish. General and Comparative Endocrinology, 1982, 48 (4), pp.462-468. 10.1016/0016-6480(82)90181-2. hal-01600561

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# Daily Variations in Plasma Cortisol Levels of Individual Female Rainbow Trout *Salmo gairdneri*: Evidence for a Post-feeding Peak in Well-Adapted Fish

C. BRY

*Laboratoire de Physiologie des Poissons, INRA 78350 Jouy en Josas, France*

Accepted January 15, 1982

Blood sampling through a catheter caused a significant and variable increase in the plasma cortisol concentration of most tested female rainbow trout. In well-adapted catheterized individuals sampled at 24- or 28-hr intervals, baseline cortisol levels were less than 8 ng/ml. A peak (15–50 ng/ml) repeatedly appeared shortly after feeding and was still present when the fish could not see the experimenter at feeding time. Other cortisol increases (12–24 ng/ml) occasionally occurred but were not reproducible. In non-feeding chronically disturbed trout, cortisol concentrations fluctuated widely (2–100 ng/ml), with no indication of periodicity.

The plasma concentration of cortisol has been used in a number of studies to evaluate the stress response of salmonid fish subjected to various traumatic procedures (see reviews by Donaldson, 1981, and Schreck, 1981). In non stressful situations, seasonal changes in cortisol or corticosteroid levels, often associated with reproduction, have been described for several salmonids (e.g., Pickering and Christie, 1981). In addition, significant fluctuations in blood levels of corticosteroid hormone within a 24-hr period have been reported in several teleost fish (review by Spieler, 1979), as well as in a large number of other vertebrate species (see, e.g., for amphibians, Dupont *et al.*, 1979; reptiles and birds, review by Barker Jorgensen, 1976; mammals, Krieger, 1979).

In contrast, some rather contradictory results have been obtained in juvenile salmonids. Baker and Rance (1980) found significant circadian changes in plasma cortisol concentrations in the immature rainbow trout (*Salmo gairdneri*). Similar studies however did not detect any marked daily fluctuations of plasma corticoid or cortisol levels in fingerling rainbow trout

(Barton *et al.*, 1980) or in other juvenile salmonids (Strange *et al.*, 1977). As the situation may be different in adult salmonids, the primary purpose of this work was to investigate plasma cortisol levels in resting adult female rainbow trout at different times of day or night.

In studies of endocrine fluctuations or rhythms, two basic methods have been used. In the majority of studies dealing with lower vertebrates and some mammals, groups of different animals have been sampled at different times. In this type of protocol, each animal is sampled once, which obviates the possible interference of stress induced by repeated handling and bleeding provided that sampling is achieved quickly. Only population variations, however, i.e., variations synchronized among individuals, can be detected with such an approach. In addition, between-group variability may be mistaken for a daily rhythm, unless replicate groups are sampled at the same time of day and during several days (Taylor *et al.*, 1978; Spieler, 1979). On the other hand, various studies, mainly related to mammals, have been using single individuals fitted with a chronic catheter and serially
sampled (e.g., Florant and Weitzman, 1980).

The use of cannulated animals appears as a desirable technique, especially when the variations of the studied parameter are not synchronized among different individuals. One, however, should consider the possible effects of cannulation and serial sampling on the physiology and behaviour of the fish. In this respect, we have recently demonstrated the possibility of obtaining well-adapted catheterized trout a few days after the operation and the nonadaptation of some fish to the presence of the catheter (Bry and Zohar, 1980). The goal of the present study was to answer the following questions:

(1) Do marked variations in plasma cortisol levels exist in well-adapted adult rainbow trout according to the time of the nycthemere, and are these variations periodic?

(2) Comparing normal and nonadapted fish: what is the influence of a chronic disturbance on the daily pattern of cortisol fluctuations?

As the handling of the catheter and the removal of blood might disturb the fish (Soivio *et al.*, 1975) and possibly interfere with the cortisol value obtained at the next sampling, a preliminary experiment was carried out to determine the short-term effects of blood sampling. Part of this work has been reported in abstract form (Bry, 1981).

## MATERIALS AND METHODS

### *Experimental Conditions*

**Experiment 1: Short-term effects of blood sampling on plasma cortisol levels and feeding activity of catheterized rainbow trout.** In both experiments, only female fish were used, in order to eliminate a further possibility of variation. In May-June, i.e., 5-7 months after the reproduction season, five 3- to 4-year-old female rainbow trout (*Salmo gairdneri* Richardson, weight range 1600-2200 g) were individually housed into rectangular tanks containing 300 liters of recycling, purified, aerated water. These fish were acclimated for 4 weeks under controlled conditions of

photoperiod (14L:10D light cycle; 0700-2100 hr light) and water temperature (13 ± 1°), and fed once a day at 1215 hr with dry pellets (1% body wt/day). Dorsal aorta catheterization was then performed and each animal was returned to its original tank. Food acceptance or refusal was noted daily at 1215 hr and the dead volume of heparinized saline was replaced once per day until Day 4. On Day 5 after cannulation and at about 1600 hr, a 0.25-ml blood sample was taken through the catheter. Blood sampling was completed by 1 min on the average, and total presence time of the experimenter averaged 2 min. Twenty minutes after the initiation of this blood collection, a second 0.25-ml sample was withdrawn under similar conditions. A few pellets were offered in succession 5 min later, and food acceptance or refusal was noted. The same experiment was also carried out with four of the females involved in experiment 2 and designated as A, C, D, and E, at the end of a sampling series.

**Experiment 2: Daily variations in plasma cortisol levels of catheterized rainbow trout.** In June-July, six adult female trout (referred to as A-F) were acclimated for 1 month in individual tanks as in experiment 1 and catheterized. Between Day 5 and Day 11 after the operation, six 0.25-ml blood samples were taken at 0830, 1230, 1630, 2030, 0030, and 0430 hr, with a 28-hr interval between successive samplings. Although the duration of the cortisol increase after blood sampling was not investigated in experiment 1, a between-sampling interval of 28 hr (rather than 4 hr) was selected for the following reasons: (1) it was felt that 28 hr would provide a better safety margin than 4 hr with regard to the possible interference of the previous sampling; (2) a 28-hr interval allowed to check between any two consecutive samplings if feeding activity was normal; (3) the hematocrit was not affected by the removal of 1.5 to 1.8 ml of blood over 7 days. The initial sampling was performed at different times according to the animal and to the sampling series (see Fig. 1-3). Food was offered daily at 1215 hr, as during the acclimation period. The 1230-hr sampling was performed 15 min after the end of the feeding session. When a fish had been noted as nonfeeding after cannulation, no feeding attempt was made on the 1230-hr sampling day, in order to eliminate a possible fright reaction due to the presence of the experimenter before sampling. In some fish (A, B, E, and F), a second series of six samples was taken under the same conditions between Day 15 and Day 21. A few additional samples were taken between Day 25 and Day 29 in fish A and B. A total of eight samples was taken from female C and seven samples from female D, including one prefeeding sample at 1215 hr and one additional postfeeding sample at 1230 hr. Some of the samples taken at 1230 hr for fish B-D were withdrawn after a feeding session during which the fish could not see the experimenter (see Figs. 1 and 2). Each trout was visually isolated from the others and no fish could see the experimenter
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FIG. 1. Daily variations in plasma cortisol levels of 2 catheterized and well-adapted female rainbow trout (A and B). Two series of six blood samples (series 1, □; series 2, ○) and a few additional samples (■) were taken from each animal, with a time interval of 28 hr or more between 2 successive samplings. The symbols □ or ○ indicate the initial sample within each series. The arrows (F) represent feeding at 1215 hr, 15 min before the next sampling (1230 hr). The symbol ◁ associated with a 1230-hr cortisol value, indicates that the fish could not see the experimenter at the corresponding feeding time (1215 hr).

sampling or feeding another fish. At night a red light with a narrow beam was used during sampling (1 min) and saline replacement (1 min) and could be detected only by the fish being sampled. No one entered the animals' quarters during the experimental periods, except for feeding or blood sampling. On a few occasions, blood clotting prevented the immediate collection of blood; in that case, the heparinized saline was replaced and blood sampling was postponed for 24 hr.

## Cortisol Assay

Each plasma sample (0.1-0.2 ml) was assayed in triplicate for cortisol, using a competitive protein binding assay adapted from Murphy (1967) and as previously described (Bry and Zohar, 1980). A chromato-

graphic step (Sephadex LH-20) adapted from Sippel *et al.* (1975) systematically preceded the assay to improve specificity. The assay had a cortisol detection limit of 2 ng/ml. The coefficients of variation were 4% (within-assay) and 9% (between-assay), for a plasma cortisol concentration of 11 ng/ml. The samples from a given animal were assayed in the same batch.

## Statistical Analyses

The cortisol levels from samples 1 and 2 (experiment 1) were compared for each animal by Welch's test after calculating a standard deviation value for each individual cortisol concentration based on the within-assay variability at the corresponding level. In experiment 2, differences between two means were determined by Student's t test, as the associated variances were found to be homogeneous.

<figure><img src="image_3.png" /></figure>

FIG. 2. Daily variations in plasma cortisol levels of 2 catheterized and well-adapted female rainbow trout (C and D), with emphasis on the levels at the time of feeding. One series of seven (C) or six (D) blood samples (□) including one prefeeding (1215 hr) and one postfeeding (1230 hr) sample was taken from each animal, as well as one additional sample at 1230 hr (■). The time interval between 2 successive samplings was 24 or 28 hr. The significance of other symbols is as indicated for Fig. 1.
<table><caption>TABLE 1
SHORT-TERM EFFECTS OF BLOOD SAMPLING ON PLASMA CORTISOL LEVELS AND FEEDING ACTIVITY OF CATHETERIZED RAINBOW TROUT<sup>a</sup></caption><thead><tr><th rowspan="2">Animal designation</th><th colspan="2">Plasma cortisol levels (ng/ml)</th><th rowspan="2">Absolute increase in cortisol concentration (ng/ml) between samples 1 and 2</th><th colspan="3">Feeding activity<sup>b</sup></th></tr><tr><th>Sample 1 (T<sub>0</sub>)</th><th>Sample 2 (T<sub>0</sub> + 20 min)</th><th>On previous days and 4 hr before sample 1</th><th>5 min after sample 2</th><th>20 hr after sample 2</th></tr></thead><tbody><tr><td>1</td><td>26</td><td>32</td><td>6</td><td>+</td><td>+</td><td>+</td></tr><tr><td>2</td><td>7</td><td>14</td><td>7</td><td>+</td><td>+</td><td>+</td></tr><tr><td>3</td><td>24</td><td>178</td><td>154</td><td>+</td><td>-</td><td>+</td></tr><tr><td>4</td><td>72</td><td>137</td><td>65</td><td>-</td><td>-</td><td>-</td></tr><tr><td>5</td><td>29</td><td>114</td><td>85</td><td>+</td><td>-</td><td>+</td></tr><tr><td>A</td><td>22</td><td>33</td><td>11</td><td>+</td><td>+</td><td>+</td></tr><tr><td>C</td><td>&lt;2</td><td>&lt;2</td><td>-</td><td>+</td><td>+</td><td>+</td></tr><tr><td>D</td><td>3</td><td>21</td><td>18</td><td>+</td><td>-</td><td>+</td></tr><tr><td>E</td><td>71</td><td>108</td><td>37</td><td>-</td><td>-</td><td>-</td></tr></tbody></table>

<sup>a</sup> Two samples of 0.25 ml were taken via the catheter 20 min apart (T₀ and T₀ + 20 min).

<sup>b</sup> +, Food acceptance; -, food refusal.

## RESULTS

### Short-Term Effects of Blood Sampling (Table 1)

The sampling of blood through the catheter, which implied the presence of the experimenter and the handling of the cannula for about 2 min induced variable reactions. Only one animal (female C) did not show any detectable increase in plasma cortisol concentration 20 min later. In the eight other fish, a significant (*P* < 0.01) increase in cortisol titers was noted. Among the fish that fed regularly on previous days and on the experimental day, some individuals (females 1, 2, A, and D) demonstrated a moderate increase in cortisol levels (+6 to +18 ng/ml) and, except for female D, feeding activity was not inhibited when tested 5 min after sample 2. In other readily feeding fish (females 3 and 5) as in females 4 and E which did not feed after cannulation, the cortisol increase was much larger (+37 to +154 ng/ml) and food pellets offered shortly after sample 2 were refused. When feeding activity was checked on the next day at 1215 hr, all fish (except 4 and E) resumed regular feeding.

### Daily Cortisol Variations in Well-Adapted Trout (Figs. 1 and 2)

The four well-adapted fish (A-D) exhibited marked fluctuations in plasma cortisol concentration according to the time of the nycthemere. Low values ranged from less than 2 to about 8 ng/ml. A cortisol peak (15-50 ng/ml; 28 ng/ml on the average) appeared in all animals at 1230 hr, 15 min after meal intake. For each fish, the mean 1230-hr level was significantly different from the mean level at other times of the nycthemere (*P* < 0.01). When the fish could not see the experimenter at feeding time (see females B-D), the postfeeding peak was unchanged (comparison between means of pooled data: *P* > 0.05). There was no evidence of cortisol increase when blood was sampled at 1215 hr, shortly before feeding time (females C and D). Other cortisol increases (12-24 ng/ml) occasionally occurred: these peaks, in comparison with the peaks associated with feeding, were of lesser (A and D) or similar (B and C) importance. When two sampling series and some additional samplings were carried out (females A and B), the postfeeding peak recurred regularly, while the other peaks
were not repeated, with the exception of the female B level at 0030 hr. The mean cortisol concentration relative to a sampling series ranged from 6 to 13 ng/ml if the 1230 hr value was included, and varied between 5 and 10 ng/ml if it was omitted.

*Daily Cortisol Variations in Nonadapted Trout (Fig. 3).*

In fish E and F, distressed by the presence of the cannula and which constantly refused food, wide variations in plasma cortisol levels were recorded. Maximum values were in the 70- to 100-ng/ml range, but cortisol titers sometimes dropped below 5 ng/ml. The mean cortisol concentration relative to a sampling series varied between 32 and 56 ng/ml. No evidence of a repro-

<figure><img src="image_4.png" /><figcaption>FIG. 3. Daily variations in plasma cortisol levels of 2 catheterized and nonadapted female rainbow trout (E and F). Two series of six blood samples (series 1, □; series 2, ○) were taken from each animal with a time interval of 28 hr or more between 2 successive samplings. The symbols □ or ○ indicate the initial sample within each series.</figcaption></figure>

ducible feature was found among the obtained patterns.

## DISCUSSION

Our data do provide clear evidence of a plasma cortisol peak, shortly after feeding, in the female rainbow trout, with an average fourfold difference between such peak and the mean level at other times of the nycthemere. This cortisol increase does not result from a human presence and it is temporary, since no longer perceptible at 1630 hr, 4 hr after meal intake. Such findings are somewhat comparable to the situation described in man, where a steep increase in plasma cortisol follows the midday meal, reaching its maximum 45 min after the beginning of the meal (e.g., Brandenberger and Follenius, 1973). In the rat, the feeding schedule is an important modulator of the plasma corticosterone rhythm and a corticosterone peak generally occurs prior to feeding (e.g., Krieger and Hauser, 1978). In contrast, trout females C and D did not show any anticipatory increase when sampled at 1215 hr, immediately before food presentation. In some fishes, meal timing entrains the activity rhythm (Nishikawa and Ishibashi, 1975) and feeding time has been shown to affect the daily pattern of corticoid variations found in groups of goldfish last fed 2 days before sampling (Delahunty et al., 1978; Spieler and Noeske, 1981). Our work is the first known report on a short-term relationship between feeding and plasma corticosteroid concentrations in fish. Whether the cortisol peak observed in the rainbow trout is due to the increased locomotion at feeding, to food ingestion or to training, has to be clarified by further investigations.

Aside from the regularly recurring post-feeding peak, the cortisol variations demonstrated in our study of well-adapted trout do not appear to follow a consistent, periodic pattern, even for a given individual. Irregular oscillations of plasma corticosteroid concentrations, superimposed on a cir-
cadian periodicity, have been established in a number of mammals (Hellman *et al*., 1970; McNatty and Thurley, 1973; Florant and Weitzman, 1980). The fluctuations observed in the trout might also be of irregular nature. The occurrence of periodical variations (other than the 1230 hr surges) cannot be ruled out, however, since we only studied six or seven different times of the nycthemere, and at 24- to 28-hr intervals: we therefore may have missed a periodical pattern, especially if this pattern was of short duration and/or not completely synchronous during successive 24-hr periods in a given individual.

Our results on the short-term effects of cannula handling and blood collection highlight the necessity to sample catheterized fish at reasonable time intervals. Similarly, the frequency of sampling did affect the corticosteroid pattern in the horse (Hoffsis *et al*., 1970). Repeated venipuncture substantially increased corticosteroid concentration in sheep, but training to blood sampling procedures reduced the effect (Bassett and Hinks, 1969). It might then be of interest to examine the feasibility of customizing fish to frequent cannula handling and blood collection.

Finally, the wide fluctuations of plasma cortisol observed in the nonfeeding fish might reflect changes in their awareness of the catheter.

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