Dietary effects of melatonin on growth performance by modulation of protein bioavailability and behavior in early weaned rats and pigs

All works related to animals were approved by the Institutional Animal Care and Use Committee (KU-2021-0015) of Korea University.

A total of twenty-four 21-day-old weaned Wistar rats were adopted for a week before the start of the experiment, and the 28-day-old rats were allotted into two experimental treatments and four replications (three rats per cage) according to their body weight ([BW] CON, basal diet; MEL, 50 ppm melatonin-supplemented diet). Rats were raised in a controlled experimental room, and the light cycle was a 12:12 light and dark cycle. The nutrient specifications of the rat feed are presented in Table 1. The BW and feed intake were measured every week, and the average daily gain (ADG), average daily feed intake (ADFI), and feed efficiency (FE) were calculated. All rats were sacrificed by CO₂ gas to collect the blood and liver samples. The weight of the liver was measured, and the relative liver weight was calculated. Whole blood was collected by the cardiac puncture method, and serum samples were obtained by centrifugation at 3,000×g and 4°C and stored at −80°C until further experiments. Hepatic tissues were collected to analyze the concentration of malondialdehyde (MDA) and the gene expression level of superoxide dismutase 3 (SOD3) and stored at −80°C until further analysis.

One hundred twenty crossbred (Landrace × Yorkshire × Duroc) weaned pigs (weaned at 21 days of age; average BW of 6.00 ± 0.15 kg) were fed for 35 days in three phase feeding programs (Day 0–7, Day 8–21, and Day 22–35). Pigs were allotted into 2 treatments with 6 replications, and each pen had 10 pigs of mixed gender. Pigs had free access to feed and water during the experimental period. The feeding program of pigs consisted of 3 phases (phase I: Days 0–7; phase II: Days 8–21; phase III: Days 22–35). The diet ingredients and specifications was shown in Table 2. Melatonin was supplemented at a dose of 50 mg in a kg of diet. All pigs were weighed weekly, and feed consumption was measured to calculate ADG, ADFI, and FE. Blood samples were collected at Days 0, 21, and 35 from the jugular vein. The collected whole blood was centrifuged at 3,000×g and 4°C, and serum samples were obtained and stored at −80°C until further analysis. The behavior of pigs was recorded using closed-circuit television (CCTV) cameras (SONY HS-652DI 1/3“CCD, NTSC 3.6 mm). The CCTV cameras were installed above the pens, and the video data were collected by a digital video recorder (DS-2008DH stand-alone DVR). All video data were analyzed by using a DVR video viewer program (MDVR Video Manager). The behavior was separated into 4 states, including resting, roaming, feeding, and fighting states, and they were calculated as the percentage of pigs per the representative days of each week (Days 3, 10, 17, 24, and 31).

Serum triglyceride (TG), blood urea nitrogen (BUN), and creatinine were measured in the rat experiment, and serum TG, total cholesterol, BUN, glucose, and creatinine were measured in the pig experiment. Briefly, plasma samples and TG standards (300 mg/dL) were loaded on 96-well microplates, and lipoprotein lipase, glycerol kinase, alpha-glycerol-3-phosphate, peroxidase (POD) and 4-aminoantipyrine (4-AAP) mixed in N-ethyl-N-m-toluidine solution were then added to the well and incubated at room temperature for 5 minutes. Optical density was measured by a colorimetric microplate reader at 550 nm wavelengths. Next, plasma samples and urea standard (30 mg/dL) were loaded on 96-well microplates, and urease mixed in sodium salicylate and sodium nitroprusside buffer solution was added to each well and incubated for 15 minutes at room temperature. After incubation, sodium hypochlorite solution was added to the well and incubated for 10 minutes at room temperature. Optical density was measured by a colorimetric microplate reader at 580 nm wavelengths. Picric acid was added to the plasma sample for deproteinization, and the reactant was centrifuged at 3000 rpm for 10 minutes. The supernatant was removed, and the CRE standard (10 mg/dL) and its diluents were loaded on a 96-well microplate in duplicate using a blank (picric acid). Optical density was measured by a colorimetric microplate reader at 515 nm wavelengths. The total cholesterol concentration was measured according to the manufacturer’s protocol. In summary, plasma samples and T-CHO standard (300 mg/dL) were loaded on 96-well microplates in duplicate, and then cholesterol esterase, cholesterol oxidase, POD and 4-AAP mixed with phenol solution were added to the well and incubated at room temperature for 5 minutes. Optical density was measured by a colorimetric microplate reader at 500 nm wavelengths. Finally, plasma samples and glucose standard (200 mg/dL) were loaded on 96-well microplates in duplicate, and in the next step, mutarotase, glucose oxidase, 4-AAP and POD mixed with phenol solution were added to the well and incubated at room temperature for 5 minutes. The optical density of the end product quinoneimine was measured by a colorimetric microplate reader at 500 nm wavelengths.

The representative indicator of lipid peroxidation, MDA, was measured by TBARS assay. Briefly, 100 mg of each liver sample was weighed, and 500 μL of distilled water was added. The samples were homogenized and centrifuged at 10,000×g for 20 min at 4°C. Then, 125 μL of supernatant was placed into a 96-well microplate, and 125 μL of TBA solution was added. After 15 and 30 hours of incubation, the optical density was measured to calculate the MDA concentration in serum.

Frozen liver samples were homogenized in TRIzol reagent (Invitrogen, Waltham, MA, USA), and the total RNA from each sample was obtained according to the manufacturer’s protocol. Subsequently, cDNA was synthesized by reverse transcription using a high-capacity cDNA synthesis kit (Applied Biosystems, Waltham, MA, USA). The relative mRNA levels of each gene were determined using StepOnePlus (Applied Biosystems) and normalized to β-actin using the 2^−ΔΔCt method. The forward primer for the SOD3 gene (5’-TCTCAGGCCTCTAGCTGGGT-3’) and reverse primer for the SOD3 gene (5’-AGGTTCCACACCTGACAAGCTC-3’) were used.

The serum concentration of melatonin was measured by GC‒MS according to Paik’s protocol [14]. Briefly, 800 μL of serum samples was mixed with 800 μL of chloroform and sonicated at 1,500×g for 10 min. After centrifugation, 600 μL of the bottom layer chloroform phase was collected, and sonication was repeated once again for bottom layer collection. The extracted sample was dried, derivatized and injected into the GC‒MS machine. The GC condition was as follows: 140°C to 315°C (15°C/min) of the oven temperature with holding 2 minutes, 300°C of detector, 250°C of injector, 1/10 of split ratio, 13 psi of pressure, 40 mL/min of purge flow, 0.5 mL/min of purge time, 1 mL/min of flow rate, helium as a carrier gas, 35 kPa of inlet pressure, 40–450 a.m.u for 1 scan/s and 70 eV for the MS detection. A capillary column was used in the specification as follows: HP-5MS, 30 m × 0.25 mm internal diameter, 0.25 µm.

All values are presented as the mean ± standard errors, and quantitative data were subjected to one-way ANOVA in SAS 9.4 software (SAS Institute, Cary, NC, USA). Multiple comparisons were performed using Duncan’s multiple range test using SAS 9.4 software (SAS Institute).

The BW of the rats were significantly higher in the melatonin-supplemented group at Days 21 and 28 than in the control group (p < 0.05); however, the ADG, ADFI, and FE during the overall experimental period were not changed by dietary melatonin supplementation (Table 3). The relative liver weight of rats fed the melatonin-supplemented diet was significantly heavier than that of rats fed the basal diet (Fig. 1A, p < 0.05). In addition, the hepatic concentration of MDA was significantly decreased by dietary melatonin supplementation (Fig. 1B, p < 0.05); however, melatonin ingestion did not change the gene expression level of SOD3 compared to that in the CON group (Fig. 1C). The concentrations of TG and creatinine were not different between the CON and MEL groups; however, the concentration of BUN was significantly lower in rats fed the melatonin-supplemented diet compared to the CON group (Figs. 2A, 2B, and 2C; p < 0.05).

Fig. 1. Liver characteristics of rats fed experimental diets at Day 35. (A) Relative liver weight, (B) concentration of malondialdehyde, and (C) gene expression level of superoxide dismutase 3. ^a,bMean values with different superscript letters were significantly different (p < 0.05). CON, basal diet; MEL, 50 ppm melatonin-supplemented diet.

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Fig. 2. Serum concentrations of biochemical nutritional markers in rats fed experimental diets at Day 35. (A) Triglyceride, (B) blood urea nitrogen, and (C) creatinine. ^a,bMean values with different superscript letters were significantly different (p < 0.05). CON, basal diet; MEL, 50 ppm melatonin-supplemented diet.

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After weaning, the BW of piglets was significantly improved at Day 28 by dietary melatonin supplementation (p < 0.05), and the final BW of pigs fed the melatonin-supplemented diet were numerically increased compared to the control group. During the whole experimental period, the ADG and ADFI were not changed by dietary melatonin supplementation, and FE was also not changed by dietary melatonin supplementation (Table 4). At Days 21 and 35, the concentration of melatonin in serum was not different as a result of dietary melatonin supplementation (Fig. 3A). Additionally, dietary melatonin supplementation did not change the concentrations of TG, total cholesterol, glucose, BUN, and creatinine between pigs fed the basal diet and those fed the melatonin-supplemented diet. However, the concentrations of TG and total cholesterol gradually decreased at Days 21 and 35 compared to the concentrations at Day 0 (Figs. 3B, 3C, 3D, 3E, and 3F). During Days 8–21, roaming behavior was significantly decreased by melatonin supplementation, and fight behavior was also significantly reduced in pigs fed the melatonin-supplemented diet compared to the basal diet group (Fig. 4A, p < 0.05). Consequently, this change led to a significant increase in resting behavior and feeding behavior in pigs fed the melatonin-supplemented diet compared to the control group during Days 8–21 (Figs. 4B and 4C, p < 0.05). Additionally, resting behavior was also significantly increased during Days 22–35 in the MEL group compared to the CON group (Fig. 4B, p < 0.05). The fight state was significantly decreased during Days 8–21 in the melatonin-supplemented group compared to the CON group (Fig. 4D, p < 0.05).

Fig. 3. Serum concentration of melatonin and biochemical nutritional markers of pigs fed experimental diets at Days 0, 21 and 35. (A) Melatonin, (B) triglyceride, (C) total cholesterol, (D) glucose, (E) blood urea nitrogen, and (F) creatinine. CON, basal diet; MEL, 50 ppm melatonin-supplemented diet.

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Fig. 4. Behavior analysis of pigs fed experimental diets during Days 0–7, 8–21, and 22–35. (A) Roaming state, (B) resting state, (C) feeding state, and (D) fight state. ^a,bMean values with different superscript letters were significantly different (p<0.05). CON, basal diet; MEL, 50 ppm melatonin-supplemented diet.

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