Animal Care and Experimental Design

Six weeks-old Wistar rats (BioLASCO, Yilan, Taiwan) were given 2 weeks to adapt to the laboratory environment and then being assigned according to their weights into four groups, namely the control group (n = 9), low dose group (1X dose, or 6.2 mL/kg of weight; n = 9), medium-dose group (2X dose, or 12.4 mL/kg of weight; n = 9), and high dose group (5X dose, or 31.0 mL/kg of weight; n = 9). During this experiment, the rat drinking water was replaced daily. Rats were given access to food and water ad libitum. The room temperature was controlled between 21 and 23°C and the humidity between 50 and 60%. The lights were switched on or off every 12 h to simulate a 12-h day-night cycle. The cage bedding was replaced twice a week, and the cages were cleaned weekly. All animal experiments performed in this study were approved by the Institutional Animal Care and Use Committee of the University of Taipei (UT108004).

As shown in Figure 1, after the rats were relocated to their respective cages (two rats per cage), they were given another week to become familiar with the cage environment and oral gavaging operation before perch essence supplementation was begun. Before all the experiments, the animals were familiarized with oral gavaging operation for at least 1 week to eliminate the possible stress interference for this procedure. The initial body weights of each group at the time of perch extract administration are shown as follow: the control group (control: 260.67 ± 1.76 g), low dose group (1X dose: 261.89 ± 2.66 g), medium-dose group (2X dose: 265.50 ± 3.92 g), and high dose group (5X dose: 270.44 ± 3.96 g). And all processes are operated by well-trained laboratory colleagues, and all animals were received the familiarization gavaging procedure for 7 days to eliminate the related stress response during acclimation period. After assigning animal treatment groups, the same gavaging operation was performed regardless of taking either placebo or treatment supplement to ensure the consistency of the experiment. The control group also received identical sham gavage as other animals with supplementation substance. We also followed the recommendation of ~10 ml/kg for each gavaging supplement in rats. The total daily amount was delivered by separating to three times, and the individual providing doses were ranged between 2.1 ml/kg (1X) and 10.3 ml/kg (5X) for each gavaging operation. Although the amount was very close to the upper limit of the recommend gavaging amount, we still tried best to minimize the possible stress during our procedure. The experimental period spanned 33 days, during which the rats received a standard chow diet (5001, PMI Nutrition International, MI, USA).

Procedure and measurement of experimental design.

In this study, the main purpose was to observe the anti-fatigue effects of fish extract to an exhaustive swimming exercise. After oral gavage feeding for 32 days, the rats were fasted for 12 h and underwent an exhaustive swimming exercise endurance test in which they were loaded with a weight equivalent to 3% of their body weight. On the day of the test, the rats began the test 60 min after receiving oral gavaging. Before and after the exhaustive swimming test, the venous blood samples were collected from the tail to measure the following circulating parameters: lactate, glucose, ammonia, thiobarbituric acid reactive substances (TBARS), corticosterone, testosterone, growth hormone, myoglobin, and blood urea nitrogen (BUN) concentrations. The rats were sacrificed immediately after the test to collect liver samples to analyze the glycogen content and perform histological staining.